Role of alveolar epithelial Early growth response-1 (Egr-1) in CD8+ T Cell mediated Lung Injury

PubMed Central

Ramana, Chilakamarti V.; Cheng, Guang-Shing; Kumar, Aseem; Kwon, Hyung- Joo; Enelow, Richard I.

2009-01-01

Influenza infection of the distal airways results in severe lung injury, a considerable portion of which is immunopathologic and attributable to the host responses. We have used a mouse model to specifically investigate the role of antiviral CD8+ T cells in this injury, and have found that the critical effector molecule is TNF-α expressed by the T cells upon antigen recognition. Interestingly, the immunopathology which ensues is characterized by significant accumulation of host inflammatory cells, recruited by chemokines expressed by the target alveolar epithelial cells. In this study we analyzed the mechanisms involved in the induction of epithelial chemokine expression triggered by antigen-specific CD8+ T cell recognition, and demonstrate that the Early growth response-1 (Egr-1) transcription factor is rapidly induced in epithelial cells, both in vitro and ex vivo, and that this is a critical regulator of a host of inflammatory chemokines. Genetic deficiency of Egr-1 significantly abrogates both the chemokine expression and the immunopathologic injury associated with T cell recognition, and it directly regulates transcriptional activity of a model CXC chemokine, MIP-2. We further demonstrate that Egr-1 induction is triggered by TNF-α– dependent ERK activation, and inhibition of this pathway ablates Egr-1 expression. These findings suggest that Egr-1 may represent an important target in mitigating the immunopathology of severe influenza infection. PMID:19786304

Local small airway epithelial injury induces global smooth muscle contraction and airway constriction

PubMed Central

Zhou, Jian; Alvarez-Elizondo, Martha B.; Botvinick, Elliot

2012-01-01

Small airway epithelial cells form a continuous sheet lining the conducting airways, which serves many functions including a physical barrier to protect the underlying tissue. In asthma, injury to epithelial cells can occur during bronchoconstriction, which may exacerbate airway hyperreactivity. To investigate the role of epithelial cell rupture in airway constriction, laser ablation was used to precisely rupture individual airway epithelial cells of small airways (<300-μm diameter) in rat lung slices (∼250-μm thick). Laser ablation of single epithelial cells using a femtosecond laser reproducibly induced airway contraction to ∼70% of the original cross-sectional area within several seconds, and the contraction lasted for up to 40 s. The airway constriction could be mimicked by mechanical rupture of a single epithelial cell using a sharp glass micropipette but not with a blunt glass pipette. These results suggest that soluble mediators released from the wounded epithelial cell induce global airway contraction. To confirm this hypothesis, the lysate of primary human small airway epithelial cells stimulated a similar airway contraction. Laser ablation of single epithelial cells triggered a single instantaneous Ca2+ wave in the epithelium, and multiple Ca2+ waves in smooth muscle cells, which were delayed by several seconds. Removal of extracellular Ca2+ or decreasing intracellular Ca2+ both blocked laser-induced airway contraction. We conclude that local epithelial cell rupture induces rapid and global airway constriction through release of soluble mediators and subsequent Ca2+-dependent smooth muscle shortening. PMID:22114176

Local small airway epithelial injury induces global smooth muscle contraction and airway constriction.

PubMed

Zhou, Jian; Alvarez-Elizondo, Martha B; Botvinick, Elliot; George, Steven C

2012-02-01

Small airway epithelial cells form a continuous sheet lining the conducting airways, which serves many functions including a physical barrier to protect the underlying tissue. In asthma, injury to epithelial cells can occur during bronchoconstriction, which may exacerbate airway hyperreactivity. To investigate the role of epithelial cell rupture in airway constriction, laser ablation was used to precisely rupture individual airway epithelial cells of small airways (<300-μm diameter) in rat lung slices (∼250-μm thick). Laser ablation of single epithelial cells using a femtosecond laser reproducibly induced airway contraction to ∼70% of the original cross-sectional area within several seconds, and the contraction lasted for up to 40 s. The airway constriction could be mimicked by mechanical rupture of a single epithelial cell using a sharp glass micropipette but not with a blunt glass pipette. These results suggest that soluble mediators released from the wounded epithelial cell induce global airway contraction. To confirm this hypothesis, the lysate of primary human small airway epithelial cells stimulated a similar airway contraction. Laser ablation of single epithelial cells triggered a single instantaneous Ca(2+) wave in the epithelium, and multiple Ca(2+) waves in smooth muscle cells, which were delayed by several seconds. Removal of extracellular Ca(2+) or decreasing intracellular Ca(2+) both blocked laser-induced airway contraction. We conclude that local epithelial cell rupture induces rapid and global airway constriction through release of soluble mediators and subsequent Ca(2+)-dependent smooth muscle shortening.

Constitutive Proteasomal Degradation of TWIST-1 in Epithelial Ovarian Cancer Stem Cells Impacts Differentiation and Metastatic Potential

PubMed Central

Yin, Gang; Alvero, Ayesha B.; Craveiro, Vinicius; Holmberg, Jennie C.; Fu, Han-Hsuan; Montagna, Michele K.; Yang, Yang; Chefetz-Menaker, Ilana; Nuti, Sudhakar; Rossi, Michael; Silasi, Dan-Arin; Rutherford, Thomas; Mor, Gil

2013-01-01

Epithelial-mesenchymal transition (EMT) is a critical process for embryogenesis but is abnormally activated during cancer metastasis and recurrence. This process enables epithelial cancer cells to acquire mobility and traits associated with stemness. It is unknown whether epithelial stem cells or epithelial cancer stem cells are able to undergo EMT, and what molecular mechanism regulates this process in these specific cell types. We found that Epithelial Ovarian Cancer Stem cells (EOC stem cells) are the source of metastatic progenitor cells through a differentiation process involving EMT and Mesenchymal-Epithelial Transition (MET). We demonstrate both in vivo and in vitro the differentiation of EOC stem cells into mesenchymal spheroid-forming cells (MSFCs) and their capacity to initiate an active carcinomatosis. Furthermore, we demonstrate that human EOC stem cells injected i.p in mice are able to form ovarian tumors, suggesting that the EOC stem cells have the ability to “home” to the ovaries and establish tumors. Most interestingly, we found that TWIST1 is constitutively degraded in EOC stem cells, and that the acquisition of TWIST1 requires additional signals that will trigger the differentiation process. These findings are relevant for understanding the differentiation and metastasis process in EOC stem cells. PMID:22349827

Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis

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

Zhang, Wenyao; Li, Xuezhong; Xu, Tong

Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferationmore » and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. - Highlights: • Inflammatory BMEs affect the properties of BMFs during mastitis. • BMEs inhibited the proliferation and promoted the migration of BMFs. • BMEs enhanced secretion of inflammatory mediators and deposition of ECM in BMFs. • Changes of the properties of BMFs were mediated by specific signal molecules.« less

Epithelial adhesion molecules and the regulation of intestinal homeostasis during neutrophil transepithelial migration

PubMed Central

Sumagin, Ronen; Parkos, Charles A

2014-01-01

Epithelial adhesion molecules play essential roles in regulating cellular function and maintaining mucosal tissue homeostasis. Some form epithelial junctional complexes to provide structural support for epithelial monolayers and act as a selectively permeable barrier separating luminal contents from the surrounding tissue. Others serve as docking structures for invading viruses and bacteria, while also regulating the immune response. They can either obstruct or serve as footholds for the immune cells recruited to mucosal surfaces. Currently, it is well appreciated that adhesion molecules collectively serve as environmental cue sensors and trigger signaling events to regulate epithelial function through their association with the cell cytoskeleton and various intracellular adapter proteins. Immune cells, particularly neutrophils (PMN) during transepithelial migration (TEM), can modulate adhesion molecule expression, conformation, and distribution, significantly impacting epithelial function and tissue homeostasis. This review discusses the roles of key intestinal epithelial adhesion molecules in regulating PMN trafficking and outlines the potential consequences on epithelial function. PMID:25838976

Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis.

PubMed

Li, Huiyan; Peng, Xuan; Wang, Yating; Cao, Shirong; Xiong, Liping; Fan, Jinjin; Wang, Yihan; Zhuang, Shougang; Yu, Xueqing; Mao, Haiping

2016-09-01

Macroautophagy/autophagy protects against cellular stress. Renal sublethal injury-triggered tubular epithelial cell cycle arrest at G2/M is associated with interstitial fibrosis. However, the role of autophagy in renal fibrosis is elusive. Here, we hypothesized that autophagy activity in tubular epithelial cells is pivotal for inhibition of cell cycle G2/M arrest and subsequent fibrogenic response. In both renal epithelial cells stimulated by angiotensin II (AGT II) and the murine kidney after unilateral ureteral obstruction (UUO), we observed that occurrence of autophagy preceded increased production of COL1 (collagen, type I). Pharmacological enhancement of autophagy by rapamycin suppressed COL1 accumulation and renal fibrosis. In contrast, genetic ablation of autophagy by proximal tubular epithelial cell-specific deletion of Atg5, with reduction of the LC3-II protein level and degradation of SQSTM1/p62, showed marked cell cycle arrest at the G2/M phase, robust COL1 deposition, and severe interstitial fibrosis in a UUO model, as compared with wild-type mice. In vitro, AGT II exposure triggered autophagy preferentially in the G1/S phase, and increased COL1 expression in the G2/M phase in renal epithelial cells. Stimulation of Atg5-deficient primary proximal tubular cells with AGT II also resulted in elevated G2/M arrest and COL1 production. Pharmacological or genetic inhibition of autophagy increased AGT II-mediated G2/M arrest. Enhanced expression of ATG5, but not the autophagy-deficient ATG5 mutant K130R, rescued the G2/M arrest, suggesting the regulation of cell cycle progression by ATG5 is autophagy dependent. In conclusion, Atg5-mediated autophagy in proximal epithelial cells is a critical host-defense mechanism that prevents renal fibrosis by blocking G2/M arrest.

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

Paquette, Stéphane G.; Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto, Ontario; Banner, David

Pandemic H1N1 influenza A (H1N1pdm) elicits stronger pulmonary inflammation than previously circulating seasonal H1N1 influenza A (sH1N1), yet mechanisms of inflammatory activation in respiratory epithelial cells during H1N1pdm infection are unclear. We investigated host responses to H1N1pdm/sH1N1 infection and virus entry mechanisms in primary human bronchial epithelial cells in vitro. H1N1pdm infection rapidly initiated a robust inflammatory gene signature (3 h post-infection) not elicited by sH1N1 infection. Protein secretion inhibition had no effect on gene induction. Infection with membrane fusion deficient H1N1pdm failed to induce robust inflammatory gene expression which was rescued with restoration of fusion ability, suggesting H1N1pdm directlymore » triggered the inflammatory signature downstream of membrane fusion. Investigation of intra-virion components revealed H1N1pdm viral RNA (vRNA) triggered a stronger inflammatory phenotype than sH1N1 vRNA. Thus, our study is first to report H1N1pdm induces greater inflammatory gene expression than sH1N1 in vitro due to direct virus–epithelial cell interaction. - Highlights: • We investigated H1N1pdm/sH1N1 infection in primary epithelial cells. • H1N1pdm directly initiated a robust inflammatory gene signature, sH1N1 did not. • H1N1pdm viral RNA triggered a stronger response than sH1N1. • H1N1pdm induces greater response due to direct virus–cell interaction. • These results have potential to impact vaccine and therapeutic development.« less

Claudin-4 is required for modulation of paracellular permeability by muscarinic acetylcholine receptor in epithelial cells.

PubMed

Cong, Xin; Zhang, Yan; Li, Jing; Mei, Mei; Ding, Chong; Xiang, Ruo-Lan; Zhang, Li-Wei; Wang, Yun; Wu, Li-Ling; Yu, Guang-Yan

2015-06-15

The epithelial cholinergic system plays an important role in water, ion and solute transport. Previous studies have shown that activation of muscarinic acetylcholine receptors (mAChRs) regulates paracellular transport of epithelial cells; however, the underlying mechanism is still largely unknown. Here, we found that mAChR activation by carbachol and cevimeline reduced the transepithelial electrical resistance (TER) and increased the permeability of paracellular tracers in rat salivary epithelial SMG-C6 cells. Carbachol induced downregulation and redistribution of claudin-4, but not occludin or ZO-1 (also known as TJP1). Small hairpin RNA (shRNA)-mediated claudin-4 knockdown suppressed, whereas claudin-4 overexpression retained, the TER response to carbachol. Mechanistically, the mAChR-modulated claudin-4 properties and paracellular permeability were triggered by claudin-4 phosphorylation through ERK1/2 (also known as MAPK3 and MAPK1, respectively). Mutagenesis assay demonstrated that S195, but not S199, S203 or S207, of claudin-4, was the target for carbachol. Subsequently, the phosphorylated claudin-4 interacted with β-arrestin2 and triggered claudin-4 internalization through the clathrin-dependent pathway. The internalized claudin-4 was further degraded by ubiquitylation. Taken together, these findings suggested that claudin-4 is required for mAChR-modulated paracellular permeability of epithelial cells through an ERK1/2, β-arrestin2, clathrin and ubiquitin-dependent signaling pathway. © 2015. Published by The Company of Biologists Ltd.

Epidermal Growth Factor Receptor Transactivation by the Cannabinoid Receptor (CB1) and Transient Receptor Potential Vanilloid 1 (TRPV1) Induces Differential Responses in Corneal Epithelial Cells

DTIC Science & Technology

2010-01-01

induced Ca2þ signaling as well as phospholipase D (PLD)-mediated phosphatidic acid formation (Islam and Akhtar, 2000; Kang et al., 2000, 2001; Mazie et...Epithelial cell motility is triggered by activation of the EGF receptor through phosphatidic acid signaling. J. Cell Sci. 119, 1645e1654. McIntosh, B.T...buffer. Cell lysates were centrifuged and supernatants were collected for measuring proteins with a bichinchoninic acid assay (BCA) protein assay kit

Zinc Transporter SLC39A7/ZIP7 Promotes Intestinal Epithelial Self-Renewal by Resolving ER Stress

PubMed Central

Ohashi, Wakana; Kimura, Shunsuke; Iwanaga, Toshihiko; Furusawa, Yukihiro; Irié, Tarou; Izumi, Hironori; Watanabe, Takashi; Hara, Takafumi; Ohara, Osamu; Koseki, Haruhiko; Sato, Toshiro; Robine, Sylvie; Mori, Hisashi; Hattori, Yuichi; Mishima, Kenji; Ohno, Hiroshi; Hase, Koji; Fukada, Toshiyuki

2016-01-01

Zinc transporters play a critical role in spatiotemporal regulation of zinc homeostasis. Although disruption of zinc homeostasis has been implicated in disorders such as intestinal inflammation and aberrant epithelial morphology, it is largely unknown which zinc transporters are responsible for the intestinal epithelial homeostasis. Here, we show that Zrt-Irt-like protein (ZIP) transporter ZIP7, which is highly expressed in the intestinal crypt, is essential for intestinal epithelial proliferation. Mice lacking Zip7 in intestinal epithelium triggered endoplasmic reticulum (ER) stress in proliferative progenitor cells, leading to significant cell death of progenitor cells. Zip7 deficiency led to the loss of Olfm4+ intestinal stem cells and the degeneration of post-mitotic Paneth cells, indicating a fundamental requirement for Zip7 in homeostatic intestinal regeneration. Taken together, these findings provide evidence for the importance of ZIP7 in maintenance of intestinal epithelial homeostasis through the regulation of ER function in proliferative progenitor cells and maintenance of intestinal stem cells. Therapeutic targeting of ZIP7 could lead to effective treatment of gastrointestinal disorders. PMID:27736879

Epstein-Barr Virus Infection of Polarized Epithelial Cells via the Basolateral Surface by Memory B Cell-Mediated Transfer Infection

PubMed Central

Shannon-Lowe, Claire; Rowe, Martin

2011-01-01

Epstein Barr virus (EBV) exhibits a distinct tropism for both B cells and epithelial cells. The virus persists as a latent infection of memory B cells in healthy individuals, but a role for infection of normal epithelial is also likely. Infection of B cells is initiated by the interaction of the major EBV glycoprotein gp350 with CD21 on the B cell surface. Fusion is triggered by the interaction of the EBV glycoprotein, gp42 with HLA class II, and is thereafter mediated by the core fusion complex, gH/gL/gp42. In contrast, direct infection of CD21-negative epithelial cells is inefficient, but efficient infection can be achieved by a process called transfer infection. In this study, we characterise the molecular interactions involved in the three stages of transfer infection of epithelial cells: (i) CD21-mediated co-capping of EBV and integrins on B cells, and activation of the adhesion molecules, (ii) conjugate formation between EBV-loaded B cells and epithelial cells via the capped adhesion molecules, and (iii) interaction of EBV glycoproteins with epithelial cells, with subsequent fusion and uptake of virions. Infection of epithelial cells required the EBV gH and gL glycoproteins, but not gp42. Using an in vitro model of normal polarized epithelia, we demonstrated that polarization of the EBV receptor(s) and adhesion molecules restricted transfer infection to the basolateral surface. Furthermore, the adhesions between EBV-loaded B cells and the basolateral surface of epithelial cells included CD11b on the B cell interacting with heparan sulphate moieties of CD44v3 and LEEP-CAM on epithelial cells. Consequently, transfer infection was efficiently mediated via CD11b-positive memory B cells but not by CD11b–negative naïve B cells. Together, these findings have important implications for understanding the mechanisms of EBV infection of normal and pre-malignant epithelial cells in vivo. PMID:21573183

Diabetes and renal tubular cell apoptosis

PubMed Central

Habib, Samy L

2013-01-01

Apoptosis contributes to the development of diabetic nephropathy, but the mechanism by which high glucose induces apoptosis is not fully understood. Apoptosis of tubular epithelial cells is a major feature of diabetic kidney disease, and hyperglycemia triggers the generation of free radicals and oxidant stress in tubular cells. Hyperglycemia and high glucose in vitro also lead to apoptosis, a form of programmed cell death. High glucose similar to those seen with hyperglycemia in people with diabetes mellitus, lead to accelerated apoptosis, a form of programmed cell death characterized by cell shrinkage, chromatin condensation and DNA fragmentation, in variety of cell types, including renal proximal tubular epithelial cells. PMID:23593533

Diabetes and renal tubular cell apoptosis.

PubMed

Habib, Samy L

2013-04-15

Apoptosis contributes to the development of diabetic nephropathy, but the mechanism by which high glucose induces apoptosis is not fully understood. Apoptosis of tubular epithelial cells is a major feature of diabetic kidney disease, and hyperglycemia triggers the generation of free radicals and oxidant stress in tubular cells. Hyperglycemia and high glucose in vitro also lead to apoptosis, a form of programmed cell death. High glucose similar to those seen with hyperglycemia in people with diabetes mellitus, lead to accelerated apoptosis, a form of programmed cell death characterized by cell shrinkage, chromatin condensation and DNA fragmentation, in variety of cell types, including renal proximal tubular epithelial cells.

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

PubMed

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

2016-11-01

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

Tissue stiffening coordinates morphogenesis by triggering collective cell migration in vivo.

PubMed

Barriga, Elias H; Franze, Kristian; Charras, Guillaume; Mayor, Roberto

2018-02-22

Collective cell migration is essential for morphogenesis, tissue remodelling and cancer invasion. In vivo, groups of cells move in an orchestrated way through tissues. This movement involves mechanical as well as molecular interactions between cells and their environment. While the role of molecular signals in collective cell migration is comparatively well understood, how tissue mechanics influence collective cell migration in vivo remains unknown. Here we investigated the importance of mechanical cues in the collective migration of the Xenopus laevis neural crest cells, an embryonic cell population whose migratory behaviour has been likened to cancer invasion. We found that, during morphogenesis, the head mesoderm underlying the cephalic neural crest stiffens. This stiffening initiates an epithelial-to-mesenchymal transition in neural crest cells and triggers their collective migration. To detect changes in their mechanical environment, neural crest cells use mechanosensation mediated by the integrin-vinculin-talin complex. By performing mechanical and molecular manipulations, we show that mesoderm stiffening is necessary and sufficient to trigger neural crest migration. Finally, we demonstrate that convergent extension of the mesoderm, which starts during gastrulation, leads to increased mesoderm stiffness by increasing the cell density underneath the neural crest. These results show that convergent extension of the mesoderm has a role as a mechanical coordinator of morphogenesis, and reveal a link between two apparently unconnected processes-gastrulation and neural crest migration-via changes in tissue mechanics. Overall, we demonstrate that changes in substrate stiffness can trigger collective cell migration by promoting epithelial-to-mesenchymal transition in vivo. More broadly, our results raise the idea that tissue mechanics combines with molecular effectors to coordinate morphogenesis.

Continual exposure to cigarette smoke extracts induces tumor-like transformation of human nontumor bronchial epithelial cells in a microfluidic chip.

PubMed

Li, Encheng; Xu, Zhiyun; Liu, Fen; Wang, Huiling; Wen, Jiabin; Shao, Shujuan; Zhang, Lichuan; Wang, Lei; Liu, Chong; Lu, Jianxin; Wang, Wenxin; Gao, Zhancheng; Wang, Qi

2014-08-01

Heavy cigarette smoking-related chronic obstructive pulmonary disease is an independent risk factor for lung squamous carcinoma. However, the mechanisms underlying the malignant transformation of bronchial epithelial cells are unclear. In our study, human tumor-adjacent bronchial epithelial cells were obtained from 10 cases with smoking-related chronic obstructive pulmonary disease and lung squamous carcinoma and cultured in an established microfluidic chip for continual exposure to cigarette smoke extracts (CSE) to investigate the potential tumor-like transformation and mechanisms. The integrated microfluidic chip included upstream concentration gradient generator and downstream cell culture chambers supplied by flowing medium containing different concentrations of CSE. Our results showed that continual exposure to low doses of CSE promoted cell proliferation whereas to high doses of CSE triggered cell apoptosis. Continual exposure to CSE promoted reactive oxygen species production in human epithelial cells in a dose-dependent manner. More importantly, continual exposure to low dose of CSE promoted the epithelial-to-mesenchymal transition process and anchorage-independent growth, and increased chromosome instability in bronchial epithelial cells, accompanied by activating the GRP78, NF-κB, and PI3K pathways. The established microfluidic chip is suitable for primary culture of human tumor-adjacent bronchial epithelial cells to investigate the malignant transformation. Continual exposure to low doses of CSE promoted tumor-like transformation of human nontumor bronchial epithelial cells by inducing reactive oxygen species production and activating the relevant signaling.

Lovastatin prevents cisplatin-induced activation of pro-apoptotic DNA damage response (DDR) of renal tubular epithelial cells

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

Krüger, Katharina; Ziegler, Verena; Hartmann, Christina

The platinating agent cisplatin (CisPt) is commonly used in the therapy of various types of solid tumors. The anticancer efficacy of CisPt largely depends on the formation of bivalent DNA intrastrand crosslinks, which stimulate mechanisms of the DNA damage response (DDR), thereby triggering checkpoint activation, gene expression and cell death. The clinically most relevant adverse effect associated with CisPt treatment is nephrotoxicity that results from damage to renal tubular epithelial cells. Here, we addressed the question whether the HMG-CoA-reductase inhibitor lovastatin affects the DDR of renal cells by employing rat renal proximal tubular epithelial (NRK-52E) cells as in vitro model.more » The data show that lovastatin has extensive inhibitory effects on CisPt-stimulated DDR of NRK-52E cells as reflected on the levels of phosphorylated ATM, Chk1, Chk2, p53 and Kap1. Mitigation of CisPt-induced DDR by lovastatin was independent of the formation of DNA damage as demonstrated by (i) the analysis of Pt-(GpG) intrastrand crosslink formation by Southwestern blot analyses and (ii) the generation of DNA strand breaks as analyzed on the level of nuclear γH2AX foci and employing the alkaline comet assay. Lovastatin protected NRK-52E cells from the cytotoxicity of high CisPt doses as shown by measuring cell viability, cellular impedance and flow cytometry-based analyses of cell death. Importantly, the statin also reduced the level of kidney DNA damage and apoptosis triggered by CisPt treatment of mice. The data show that the lipid-lowering drug lovastatin extensively counteracts pro-apoptotic signal mechanisms of the DDR of tubular epithelial cells following CisPt injury. - Highlights: • Lovastatin blocks ATM/ATR-regulated DDR of tubular cells following CisPt treatment. • Lovastatin attenuates CisPt-induced activation of protein kinase ATM in vitro. • Statin-mediated DDR inhibition is independent of initial DNA damage formation. • Statin-mediated blockage of CisPt-triggered DDR leads to cytoprotection. • Lovastatin attenuates CisPt-induced kidney DNA damage and apoptosis in vivo.« less

Mechanical strain induces involution-associated events in mammary epithelial cells

PubMed Central

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

2009-01-01

Background Shortly after weaning, a complex multi-step process that leads to massive epithelial apoptosis is triggered by tissue local factors in the mouse mammary gland. Several reports have demonstrated the relevance of mechanical stress to induce adaptive responses in different cell types. Interestingly, these signaling pathways also participate in mammary gland involution. Then, it has been suggested that cell stretching caused by milk accumulation after weaning might be the first stimulus that initiates the complete remodeling of the mammary gland. However, no previous report has demonstrated the impact of mechanical stress on mammary cell physiology. To address this issue, we have designed a new practical device that allowed us to evaluate the effects of radial stretching on mammary epithelial cells in culture. Results We have designed and built a new device to analyze the biological consequences of applying mechanical stress to cells cultured on flexible silicone membranes. Subsequently, a geometrical model that predicted the percentage of radial strain applied to the elastic substrate was developed. By microscopic image analysis, the adjustment of these calculations to the actual strain exerted on the attached cells was verified. The studies described herein were all performed in the HC11 non-tumorigenic mammary epithelial cell line, which was originated from a pregnant BALB/c mouse. In these cells, as previously observed in other tissue types, mechanical stress induced ERK1/2 phosphorylation and c-Fos mRNA and protein expression. In addition, we found that mammary cell stretching triggered involution associated cellular events as Leukemia Inhibitory Factor (LIF) expression induction, STAT3 activation and AKT phosphorylation inhibition. Conclusion Here, we show for the first time, that mechanical strain is able to induce weaning-associated events in cultured mammary epithelial cells. These results were obtained using a new practical and affordable device specifically designed for such a purpose. We believe that our results indicate the relevance of mechanical stress among the early post-lactation events that lead to mammary gland involution. PMID:19615079

Cell-Specific Imd-NF-κB Responses Enable Simultaneous Antibacterial Immunity and Intestinal Epithelial Cell Shedding upon Bacterial Infection.

PubMed

Zhai, Zongzhao; Boquete, Jean-Philippe; Lemaitre, Bruno

2018-05-03

Intestinal infection triggers potent immune responses to combat pathogens and concomitantly drives epithelial renewal to maintain barrier integrity. Current models propose that epithelial renewal is primarily driven by damage caused by reactive oxygen species (ROS). Here we found that in Drosophila, the Imd-NF-κB pathway controlled enterocyte (EC) shedding upon infection, via a mechanism independent of ROS-associated apoptosis. Mechanistically, the Imd pathway synergized with JNK signaling to induce epithelial cell shedding specifically in the context of bacterial infection, requiring also the reduced expression of the transcription factor GATAe. Furthermore, cell-specific NF-κB responses enabled simultaneous production of antimicrobial peptides (AMPs) and epithelial shedding in different EC populations. Thus, the Imd-NF-κB pathway is central to the intestinal antibacterial response by mediating both AMP production and the maintenance of barrier integrity. Considering the similarities between Drosophila Imd signaling and mammalian TNFR pathway, our findings suggest the existence of an evolutionarily conserved genetic program in immunity-induced epithelial shedding. Copyright © 2018 Elsevier Inc. All rights reserved.

AKT-ions with a TWIST between EMT and MET.

PubMed

Tang, Huifang; Massi, Daniela; Hemmings, Brian A; Mandalà, Mario; Hu, Zhengqiang; Wicki, Andreas; Xue, Gongda

2016-09-20

The transcription factor Twist is an important regulator of cranial suture during embryogenesis. Closure of the neural tube is achieved via Twist-triggered cellular transition from an epithelial to mesenchymal phenotype, a process known as epithelial-mesenchymal transition (EMT), characterized by a remarkable increase in cell motility. In the absence of Twist activity, EMT and associated phenotypic changes in cell morphology and motility can also be induced, albeit moderately, by other transcription factor families, including Snail and Zeb. Aberrant EMT triggered by Twist in human mammary tumour cells was first reported to drive metastasis to the lung in a metastatic breast cancer model. Subsequent analysis of many types of carcinoma demonstrated overexpression of these unique EMT transcription factors, which statistically correlated with worse outcome, indicating their potential as biomarkers in the clinic. However, the mechanisms underlying their activation remain unclear. Interestingly, increasing evidence indicates they are selectively activated by distinct intracellular kinases, thereby acting as downstream effectors facilitating transduction of cytoplasmic signals into nucleus and reprogramming EMT and mesenchymal-epithelial transition (MET) transcription to control cell plasticity. Understanding these relationships and emerging data indicating differential phosphorylation of Twist leads to complex and even paradoxical functionalities, will be vital to unlocking their potential in clinical settings.

Romo1 expression contributes to oxidative stress-induced death of lung epithelial cells

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

Shin, Jung Ar; Chung, Jin Sil; Cho, Sang-Ho

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.more » 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.« less

Cigarette smoke induced urocystic epithelial mesenchymal transition via MAPK pathways.

PubMed

Yu, Dexin; Geng, Hao; Liu, Zhiqi; Zhao, Li; Liang, Zhaofeng; Zhang, Zhiqiang; Xie, Dongdong; Wang, Yi; Zhang, Tao; Min, Jie; Zhong, Caiyun

2017-01-31

Cigarette smoke has been shown to be a major risk factor for bladder cancer. Epithelial-mesenchymal transition (EMT) is a crucial process in cancer development. The role of MAPK pathways in regulating cigarette smoke-triggered urocystic EMT remains to be elucidated. Human normal urothelial cells and BALB/c mice were used as in vitro and in vivo cigarette smoke exposure models. Exposure of human normal urothelial cells to cigarette smoke induced morphological change, enhanced migratory and invasive capacities, reduced epithelial marker expression and increased mesenchymal marker expression, along with the activation of MAPK pathways. Moreover, we revealed that ERK1/2 and p38 inhibitors, but rather JNK inhibitor, effectively attenuated cigarette smoke-induced urocystic EMT. Importantly, the regulatory function of ERK1/2 and p38 pathways in cigarette smoke-triggered urocystic EMT was further confirmed in mice exposed to CS for 12 weeks. These findings could provide new insight into the molecular mechanisms of cigarette smoke-associated bladder cancer development as well as its potential intervention.

The Epithelial Cell-derived Atopic Dermatitis Cytokine TSLP Activates Neurons to Induce Itch

PubMed Central

Wilson, Sarah R.; Thé, Lydia; Batia, Lyn M.; Beattie, Katherine; Katibah, George E.; McClain, Shannan P.; Pellegrino, Maurizio; Estandian, Daniel M.; Bautista, Diana M.

2014-01-01

Summary Atopic dermatitis (AD) is a chronic itch and inflammatory disorder of the skin that affects one in ten people. Patients suffering from severe AD eventually progress to develop asthma and allergic rhinitis, in a process known as the “atopic march.” Signaling between epithelial cells and innate immune cells via the cytokine Thymic Stromal Lymphopoietin (TSLP) is thought to drive AD and the atopic march. Here we report that epithelial cells directly communicate to cutaneous sensory neurons via TSLP to promote itch. We identify the ORAI1/NFAT calcium signaling pathway as an essential regulator of TSLP release from keratinocytes, the primary epithelial cells of the skin. TSLP then acts directly on a subset of TRPA1-positive sensory neurons to trigger robust itch behaviors. Our results support a new model whereby calcium-dependent TSLP release by keratinocytes activates both primary afferent neurons and immune cells to promote inflammatory responses in the skin and airways. PMID:24094650

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

PubMed

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

2014-10-15

Transdifferentiation of epithelial cells into mesenchymal cells and myofibroblasts plays an important role in tumor progression and tissue fibrosis. Such epithelial plasticity is accompanied by dramatic reorganizations of the actin cytoskeleton, although mechanisms underlying cytoskeletal effects on epithelial transdifferentiation remain poorly understood. In the present study, we observed that selective siRNA-mediated knockdown of γ-cytoplasmic actin (γ-CYA), but not β-cytoplasmic actin, induced epithelial-to-myofibroblast transition (EMyT) of different epithelial cells. The EMyT manifested by increased expression of α-smooth muscle actin and other contractile proteins, along with inhibition of genes responsible for cell proliferation. Induction of EMyT in γ-CYA-depleted cells depended on activation of serum response factor and its cofactors, myocardial-related transcriptional factors A and B. Loss of γ-CYA stimulated formin-mediated actin polymerization and activation of Rho GTPase, which appear to be essential for EMyT induction. Our findings demonstrate a previously unanticipated, unique role of γ-CYA in regulating epithelial phenotype and suppression of EMyT that may be essential for cell differentiation and tissue fibrosis. © 2014 Lechuga, Baranwal, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Secreted Oral Epithelial Cell Membrane Vesicles Induce Epstein-Barr Virus Reactivation in Latently Infected B Cells

PubMed Central

Lin, Zhen; Swan, Kenneth; Zhang, Xin; Cao, Subing; Brett, Zoe; Drury, Stacy; Fewell, Claire; Puetter, Adriane; Wang, Xia; Ferris, MaryBeth; Sullivan, Deborah E.; Li, Li

2016-01-01

ABSTRACT In the oral epithelium, peripheral stores of Epstein-Barr virus (EBV) are transmitted from infiltrating B cells to epithelial cells. Once the virus is transmitted to epithelial cells, the highly permissive nature of this cell type for lytic replication allows virus amplification and exchange to other hosts. Since the initial transfer of EBV from B cells to epithelial cells requires transitioning of the B-cell to a state that induces virus reactivation, we hypothesized that there might be epithelium-specific signals that allow the infiltrating B cells to sense the appropriate environment to initiate reactivation and begin this exchange process. We previously found that the epithelium-specific miR-200 family of microRNAs promotes EBV lytic replication. Here we show that there are high levels of miR-200 family members in oral and tonsillar epithelia and in saliva. Analysis of cultured oral epithelial cells (OKF6) showed that they actively secrete membrane vesicles (exosomes) that are enriched with miR-200 family members. Coculturing of EBV-positive B cells with OKF6 cells induced viral reactivation. Further, treatment of EBV-positive B cells with OKF6 cell-derived membrane vesicles promoted reactivation. Using a cell system that does not naturally express miR-200 family members, we found that enforced expression of a miR-200 family member produced membrane vesicles that were able to induce the lytic cascade in EBV-positive B cells. We propose that membrane vesicles secreted by oral and tonsillar epithelial cells may serve as a tissue-specific environmental cue that initiates reactivation in B cells, promoting the transfer of virus from peripheral B-cell stores to the oral epithelium to facilitate virus amplification and exchange to other hosts. IMPORTANCE Epstein-Barr virus (EBV) is an important human pathogen that is causally associated with several lymphomas and carcinomas. The switch from latency to the lytic cycle is critical for successful host infection and for EBV pathogenesis. Although the EBV lytic cycle can be triggered by certain agents in vitro, the mechanisms that signal reactivation in vivo are poorly understood. We previously reported that endogenously expressed miR-200 family members likely play a role in facilitating the lytic tendencies of EBV in epithelial cells. Here we show that membrane vesicles secreted from oral epithelial cells contain miR-200 family members and that they can be transmitted to proximal EBV-positive B cells, where they trigger reactivation. We propose that this intercellular communication pathway may serve as a sensor mechanism for infiltrating B cells to recognize an appropriate environment to initiate reactivation, thereby allowing the exchange of virus to the oral epithelium. PMID:26764001

Copper Uptake in Mammary Epithelial Cells Activates Cyclins and Triggers Antioxidant Response

PubMed Central

dos Santos, Nathália Villa; Matias, Andreza Cândido; Higa, Guilherme Shigueto Vilar; Kihara, Alexandre Hiroaki; Cerchiaro, Giselle

2015-01-01

The toxicologic effects of copper (Cu) on tumor cells have been studied during the past decades, and it is suggested that Cu ion may trigger antiproliferative effects in vitro. However, in normal cells the toxicologic effects of high exposures of free Cu are not well understood. In this work, Cu uptake, the expression of genes associated with cell cycle regulation, and the levels of ROS production and related oxidative processes were evaluated in Cu-treated mammary epithelial MCF10A nontumoral cells. We have shown that the Cu additive is associated with the activation of cyclin D1 and cyclin B1, as well as cyclin-dependent kinase 2 (CDK2). These nontumor cells respond to Cu-induced changes in the oxidative balance by increase of the levels of reduced intracellular glutathione (GSH), decrease of reactive oxygen species (ROS) generation, and accumulation during progression of the cell cycle, thus preventing the cell abnormal proliferation or death. Taken together, our findings revealed an effect that contributes to prevent a possible damage of normal cells exposed to chemotherapeutic effects of drugs containing the Cu ion. PMID:26583055

Epithelial-mesenchymal transition in tissue repair and fibrosis.

PubMed

Stone, Rivka C; Pastar, Irena; Ojeh, Nkemcho; Chen, Vivien; Liu, Sophia; Garzon, Karen I; Tomic-Canic, Marjana

2016-09-01

The epithelial-mesenchymal transition (EMT) describes the global process by which stationary epithelial cells undergo phenotypic changes, including the loss of cell-cell adhesion and apical-basal polarity, and acquire mesenchymal characteristics that confer migratory capacity. EMT and its converse, MET (mesenchymal-epithelial transition), are integral stages of many physiologic processes and, as such, are tightly coordinated by a host of molecular regulators. Converging lines of evidence have identified EMT as a component of cutaneous wound healing, during which otherwise stationary keratinocytes (the resident skin epithelial cells) migrate across the wound bed to restore the epidermal barrier. Moreover, EMT plays a role in the development of scarring and fibrosis, as the matrix-producing myofibroblasts arise from cells of the epithelial lineage in response to injury but are pathologically sustained instead of undergoing MET or apoptosis. In this review, we summarize the role of EMT in physiologic repair and pathologic fibrosis of tissues and organs. We conclude that further investigation into the contribution of EMT to the faulty repair of fibrotic wounds might identify components of EMT signaling as common therapeutic targets for impaired healing in many tissues. Graphical Abstract Model for injury-triggered EMT activation in physiologic wound repair (left) and fibrotic wound healing (right).

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

PubMed Central

Xiong, Jimin; Mrozik, Krzysztof; Gronthos, Stan

2012-01-01

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

Pathogenesis of human diffusely adhering Escherichia coli expressing Afa/Dr adhesins (Afa/Dr DAEC): current insights and future challenges.

PubMed

Servin, Alain L

2014-10-01

The pathogenicity and clinical pertinence of diffusely adhering Escherichia coli expressing the Afa/Dr adhesins (Afa/Dr DAEC) in urinary tract infections (UTIs) and pregnancy complications are well established. In contrast, the implication of intestinal Afa/Dr DAEC in diarrhea is still under debate. These strains are age dependently involved in diarrhea in children, are apparently not involved in diarrhea in adults, and can also be asymptomatic intestinal microbiota strains in children and adult. This comprehensive review analyzes the epidemiology and diagnosis and highlights recent progress which has improved the understanding of Afa/Dr DAEC pathogenesis. Here, I summarize the roles of Afa/Dr DAEC virulence factors, including Afa/Dr adhesins, flagella, Sat toxin, and pks island products, in the development of specific mechanisms of pathogenicity. In intestinal epithelial polarized cells, the Afa/Dr adhesins trigger cell membrane receptor clustering and activation of the linked cell signaling pathways, promote structural and functional cell lesions and injuries in intestinal barrier, induce proinflammatory responses, create angiogenesis, instigate epithelial-mesenchymal transition-like events, and lead to pks-dependent DNA damage. UTI-associated Afa/Dr DAEC strains, following adhesin-membrane receptor cell interactions and activation of associated lipid raft-dependent cell signaling pathways, internalize in a microtubule-dependent manner within urinary tract epithelial cells, develop a particular intracellular lifestyle, and trigger a toxin-dependent cell detachment. In response to Afa/Dr DAEC infection, the host epithelial cells generate antibacterial defense responses. Finally, I discuss a hypothetical role of intestinal Afa/Dr DAEC strains that can act as "silent pathogens" with the capacity to emerge as "pathobionts" for the development of inflammatory bowel disease and intestinal carcinogenesis. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Clonidine Induces Apoptosis of Human Corneal Epithelial Cells through Death Receptors-Mediated, Mitochondria-Dependent Signaling Pathway.

PubMed

Fan, Dan; Fan, Ting-Jun

2017-03-01

Clonidine, an α2-adrenoreceptor agonist, is an anti-glaucoma drug clinically used in many developing countries, and its abuse might damage the cornea and impair human vision. However, its cytotoxicity and precise mechanisms need to be elucidated. Herein, we investigated the cytotoxicity of clonidine and its underlying mechanisms, using an in vitro model of human corneal epithelial (HCEP) cells and an in vivo model of cat corneas, respectively. HCEP cells were treated with various doses of clonidine for 1-28 h, resulting in abnormal morphology, decline of cell viability and G1 phase arrest in a time- and/or dose-dependent manner. Moreover, clonidine treatment induced elevation of plasma membrane permeability, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation in HCEP cells. Furthermore, we found that clonidine treatment resulted in activated caspase-2, -3, -8, and -9, disruption of the mitochondrial transmembrane potential, downregulation of Bcl-2, and upregulation of Bad, cytoplasmic cytochrome c and apoptosis inducing factor, suggesting that clonidine-induced apoptosis is triggered through Fas/TNFR1 death receptors and Bcl-2 family proteins-mediated mitochondria-dependent pathways. Finally, our in vivo results displayed that 0.25% clonidine could induce DNA fragmentation of cat corneal epithelial cells. In summary, our findings suggest that clonidine above 1/32 of its clinical therapeutic dosage is cytotoxic to corneal epithelial cells by inducing cell apoptosis both in vitro and in vivo, and its pro-apoptotic effect on HCEP cells is triggered by a Fas/TNFR1 death receptors-mediated, mitochondria-dependent signaling pathway. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Pathogenesis of Human Diffusely Adhering Escherichia coli Expressing Afa/Dr Adhesins (Afa/Dr DAEC): Current Insights and Future Challenges

PubMed Central

2014-01-01

SUMMARY The pathogenicity and clinical pertinence of diffusely adhering Escherichia coli expressing the Afa/Dr adhesins (Afa/Dr DAEC) in urinary tract infections (UTIs) and pregnancy complications are well established. In contrast, the implication of intestinal Afa/Dr DAEC in diarrhea is still under debate. These strains are age dependently involved in diarrhea in children, are apparently not involved in diarrhea in adults, and can also be asymptomatic intestinal microbiota strains in children and adult. This comprehensive review analyzes the epidemiology and diagnosis and highlights recent progress which has improved the understanding of Afa/Dr DAEC pathogenesis. Here, I summarize the roles of Afa/Dr DAEC virulence factors, including Afa/Dr adhesins, flagella, Sat toxin, and pks island products, in the development of specific mechanisms of pathogenicity. In intestinal epithelial polarized cells, the Afa/Dr adhesins trigger cell membrane receptor clustering and activation of the linked cell signaling pathways, promote structural and functional cell lesions and injuries in intestinal barrier, induce proinflammatory responses, create angiogenesis, instigate epithelial-mesenchymal transition-like events, and lead to pks-dependent DNA damage. UTI-associated Afa/Dr DAEC strains, following adhesin-membrane receptor cell interactions and activation of associated lipid raft-dependent cell signaling pathways, internalize in a microtubule-dependent manner within urinary tract epithelial cells, develop a particular intracellular lifestyle, and trigger a toxin-dependent cell detachment. In response to Afa/Dr DAEC infection, the host epithelial cells generate antibacterial defense responses. Finally, I discuss a hypothetical role of intestinal Afa/Dr DAEC strains that can act as “silent pathogens” with the capacity to emerge as “pathobionts” for the development of inflammatory bowel disease and intestinal carcinogenesis. PMID:25278576

Reovirus-Induced Apoptosis in the Intestine Limits Establishment of Enteric Infection.

PubMed

Brown, Judy J; Short, Sarah P; Stencel-Baerenwald, Jennifer; Urbanek, Kelly; Pruijssers, Andrea J; McAllister, Nicole; Ikizler, Mine; Taylor, Gwen; Aravamudhan, Pavithra; Khomandiak, Solomiia; Jabri, Bana; Williams, Christopher S; Dermody, Terence S

2018-05-15

Several viruses induce intestinal epithelial cell death during enteric infection. However, it is unclear whether proapoptotic capacity promotes or inhibits replication in this tissue. We infected mice with two reovirus strains that infect the intestine but differ in the capacity to alter immunological tolerance to new food antigen. Infection with reovirus strain T1L, which induces an inflammatory immune response to fed antigen, is prolonged in the intestine, whereas T3D-RV, which does not induce this response, is rapidly cleared from the intestine. Compared with T1L, T3D-RV infection triggered apoptosis of intestinal epithelial cells and subsequent sloughing of dead cells into the intestinal lumen. We conclude that the infection advantage of T1L derives from its capacity to subvert host restriction by epithelial cell apoptosis, providing a possible mechanism by which T1L enhances inflammatory signals during antigen feeding. Using a panel of T1L × T3D-RV reassortant viruses, we identified the viral M1 and M2 gene segments as determinants of reovirus-induced apoptosis in the intestine. Expression of the T1L M1 and M2 genes in a T3D-RV background was sufficient to limit epithelial cell apoptosis and enhance viral infection to levels displayed by T1L. These findings define additional reovirus gene segments required for enteric infection of mice and illuminate the antiviral effect of intestinal epithelial cell apoptosis in limiting enteric viral infection. Viral strain-specific differences in the capacity to infect the intestine may be useful in identifying viruses capable of ameliorating tolerance to fed antigen in autoimmune conditions like celiac disease. IMPORTANCE Acute viral infections are thought to be cleared by the host with few lasting consequences. However, there may be much broader and long-lasting effects of viruses on immune homeostasis. Infection with reovirus, a common, nonpathogenic virus, triggers inflammation against innocuous food antigens, implicating this virus in the development of celiac disease, an autoimmune intestinal disorder triggered by exposure to dietary gluten. Using two reovirus strains that differ in the capacity to abrogate oral tolerance, we found that strain-specific differences in the capacity to replicate in the intestine inversely correlate with the capacity to induce apoptotic death of intestinal epithelial cells, providing a host-mediated process to restrict intestinal infection. This work contributes new knowledge about virus-host interactions in the intestine and establishes a foundation for future studies to define mechanisms by which viruses break oral tolerance in celiac disease. Copyright © 2018 American Society for Microbiology.

The adaptor protein Cindr regulates JNK activity to maintain epithelial sheet integrity.

PubMed

Yasin, Hannah W R; van Rensburg, Samuel H; Feiler, Christina E; Johnson, Ruth I

2016-02-15

Epithelia are essential barrier tissues that must be appropriately maintained for their correct function. To achieve this a plethora of protein interactions regulate epithelial cell number, structure and adhesion, and differentiation. Here we show that Cindr (the Drosophila Cin85 and Cd2ap ortholog) is required to maintain epithelial integrity. Reducing Cindr triggered cell delamination and movement. Most delaminating cells died. These behaviors were consistent with JNK activation previously associated with loss of epithelial integrity in response to ectopic oncogene activity. We confirmed a novel interaction between Cindr and Drosophila JNK (dJNK), which when perturbed caused inappropriate JNK signaling. Genetically reducing JNK signaling activity suppressed the effects of reducing Cindr. Furthermore, ectopic JNK signaling phenocopied loss of Cindr and was partially rescued by concomitant cindr over-expression. Thus, correct Cindr-dJNK stoichiometry is essential to maintain epithelial integrity and disturbing this balance may contribute to the pathogenesis of disease states, including cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

CCAAT/enhancer binding protein beta (C/EBPβ) isoform balance as a regulator of epithelial-mesenchymal transition in mouse mammary epithelial cells

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

Miura, Yuka; Hagiwara, Natsumi; Radisky, Derek C.

2014-09-10

Activation of the epithelial-mesenchymal transition (EMT) program promotes cell invasion and metastasis, and is reversed through mesenchymal-epithelial transition (MET) after formation of distant metastases. Here, we show that an imbalance of gene products encoded by the transcriptional factor C/EBPβ, LAP (liver-enriched activating protein) and LIP (liver-enriched inhibitory protein), can regulate both EMT- and MET-like phenotypic changes in mouse mammary epithelial cells. By using tetracycline repressive LIP expression constructs, we found that SCp2 cells, a clonal epithelial line of COMMA1-D cells, expressed EMT markers, lost the ability to undergo alveolar-like morphogenesis in 3D Matrigel, and acquired properties of benign adenoma cells.more » Conversely, we found that inducible expression of LAP in SCg6 cells, a clonal fibroblastic line of COMMA1-D cells, began to express epithelial keratins with suppression of proliferation. The overexpression of the C/EBPβ gene products in these COMMA1-D derivatives was suppressed by long-term cultivation on tissue culture plastic, but gene expression was maintained in cells grown on Matrigel or exposed to proteasome inhibitors. Thus, imbalances of C/EBPβ gene products in mouse mammary epithelial cells, which are affected by contact with basement membrane, are defined as a potential regulator of metastatic potential. - Highlights: • We created a temporal imbalance of C/EBPβ gene products in the mammary model cells. • The temporal up-regulation of LIP protein induced EMT-like cell behaviors. • The temporal up-regulation of LAP protein induced MET-like cell behaviors. • Excess amount of C/EBPβ gene products were eliminated by proteasomal-degradation. • Basement membrane components attenuated proteasome-triggered protein elimination.« less

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

PubMed Central

Sakai, Norihiko; Tager, Andrew M.

2013-01-01

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

Ascites-induced shift along epithelial-mesenchymal spectrum in ovarian cancer cells: enhancement of their invasive behavior partly dependant on αv integrins.

PubMed

Carduner, L; Leroy-Dudal, J; Picot, C R; Gallet, O; Carreiras, F; Kellouche, S

2014-08-01

At least one-third of patients with epithelial ovarian cancer (OC) present ascites at diagnosis and almost all have ascites at recurrence. The presence of ascites, which acts as a dynamic reservoir of active molecules and cellular components, correlates with the OC peritoneal metastasis and is associated with poor prognosis. Since epithelial-mesenchymal transition (EMT) is involved in different phases of OC progression, we have investigated the effect of the unique ascitic tumor microenvironment on the EMT status and the behavior of OC cells. The exposure of three OC cell lines to ascites leads to changes in cellular morphologies. Within ascites, OC cells harboring an initial intermediate epithelial phenotype are characterized by marked dislocation of epithelial markers (E-cadherin, ZO-1 staining) while OC cells initially harboring an intermediate mesenchymal phenotype strengthen their mesenchymal markers (N-cadherin, vimentin). Ascites differentially triggers a dissemination phenotype related to the initial cell features by either allowing the proliferation and the formation of spheroids and the extension of colonies for cells that present an initial epithelial intermediate phenotype, or favoring the migration of cells with a mesenchymal intermediate phenotype. In an ascitic microenvironment, a redeployment of αv integrins into cells was observed and the ascites-induced accentuation of the two different invasive phenotypes (i.e. spheroids formation or migration) was shown to involve αv integrins. Thus, ascites induces a shift toward an unstable intermediate state of the epithelial-mesenchymal spectrum and confers a more aggressive cell behavior that takes on a different pathway based on the initial epithelial-mesenchymal cell features.

EphA2 Drives the Segregation of Ras-Transformed Epithelial Cells from Normal Neighbors.

PubMed

Porazinski, Sean; de Navascués, Joaquín; Yako, Yuta; Hill, William; Jones, Matthew Robert; Maddison, Robert; Fujita, Yasuyuki; Hogan, Catherine

2016-12-05

In epithelial tissues, cells expressing oncogenic Ras (hereafter RasV12 cells) are detected by normal neighbors and as a result are often extruded from the tissue [1-6]. RasV12 cells are eliminated apically, suggesting that extrusion may be a tumor-suppressive process. Extrusion depends on E-cadherin-based cell-cell adhesions and signaling to the actin-myosin cytoskeleton [2, 6]. However, the signals underlying detection of the RasV12 cell and triggering extrusion are poorly understood. Here we identify differential EphA2 signaling as the mechanism by which RasV12 cells are detected in epithelial cell sheets. Cell-cell interactions between normal cells and RasV12 cells trigger ephrin-A-EphA2 signaling, which induces a cell repulsion response in RasV12 cells. Concomitantly, RasV12 cell contractility increases in an EphA2-dependent manner. Together, these responses drive the separation of RasV12 cells from normal cells. In the absence of ephrin-A-EphA2 signals, RasV12 cells integrate with normal cells and adopt a pro-invasive morphology. We also show that Drosophila Eph (DEph) is detected in segregating clones of RasV12 cells and is functionally required to drive segregation of RasV12 cells in vivo, suggesting that our in vitro findings are conserved in evolution. We propose that expression of RasV12 in single or small clusters of cells within a healthy epithelium creates ectopic EphA2 boundaries, which drive the segregation and elimination of the transformed cell from the tissue. Thus, deregulation of Eph/ephrin would allow RasV12 cells to go undetected and expand within an epithelium. Copyright © 2016 Elsevier Ltd. All rights reserved.

Use of Methacrylic Acid-Containing Hydrogels to Increase Protein Transport Across the Intestinal Epithelium

NASA Astrophysics Data System (ADS)

Blanchette, James; Lopez, Jennifer; Park, Kinam; Peppas, Nicholas

2002-03-01

Oral protein delivery requires protection from the harsh environment of the stomach, release in the small intestine and passage from the intestinal lumen into the circulation. Hydrogels that swell in response to the pH change when passing from the stomach to the small intestine can accomplish the first two points. The ability to enhance the permeability of intestinal epithelial cells is currently under investigation. Methacrylic acid-containing hydrogels have shown the ability to bind calcium ions that decreases the concentration of free extracellular calcium for these epithelial cells. This change triggers a number of intracellular events including rearrangement of the cytoskeleton leading to increased permeability. Studies done on Caco-2 cells (human colon adenocarcinoma) measuring changes in transepithelial resistance are used to assess the effect of the polymer-cell interactions on the integrity of intestinal epithelial cell monolayers.

Role of Candida albicans polymorphism in interactions with oral epithelial cells.

PubMed

Villar, C C; Kashleva, H; Dongari-Bagtzoglou, A

2004-08-01

Candida albicans is a polymorphic organism which undergoes morphologic transition between yeast, pseudohyphal and hyphal forms. The ability of C. albicans to change from yeast to filamentous types is a major virulence determinant of this organism. However, the exact role of hyphal transformation in establishing oral mucosal infection is still poorly understood. In this study we used mutants with defects in filamentation, as well as oral strains, which differ in their capacity to form true hyphae, to examine the role of hyphal transformation in the interactions of C. albicans with oral epithelial cells in vitro. These interactions included the ability of these strains to adhere to and injure epithelial cells, as well as their ability to trigger a proinflammatory cytokine response. We found that strains SC5314 and ATCC28366 formed true hyphae on epithelial cells, whereas strain ATCC32077 and the tup1/tup1 mutant formed only pseudohyphae. Double mutant efg1/efg1cph1/cph1 grew exclusively as blastospores. We also found that yeast and pseudohyphal strains showed reduced adherence capacity to oral keratinocytes and caused minimal cell damage. Moreover, we showed that both yeast and pseudohyphal forms have a strongly attenuated proinflammatory phenotype, since they failed to induce significant interleukin (IL)-1alpha and IL-8 responses by oral epithelial cells. Germination of C. albicans into true hyphae is particularly important in the interactions with oral epithelial cells in vitro.

Deciliation Is Associated with Dramatic Remodeling of Epithelial Cell Junctions and Surface Domains

PubMed Central

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

2009-01-01

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

Serratia marcescens internalization and replication in human bladder epithelial cells

PubMed Central

Hertle, Ralf; Schwarz, Heinz

2004-01-01

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

The stress polarity pathway: AMPK ‘GIV’-es protection against metabolic insults

PubMed Central

Ghosh, Pradipta

2017-01-01

Loss of cell polarity impairs organ development and function; it can also serve as one of the first triggers for oncogenesis. In 2006-2007 two groups simultaneously reported the existence of a special pathway for maintaining epithelial polarity in the face of environmental stressors. In this pathway, AMPK, a key sensor of metabolic stress stabilizes tight junctions, preserves cell polarity, and thereby, maintains epithelial barrier functions. Accumulating evidence since has shown that pharmacologic activation of AMPK by Metformin protects the epithelial barrier against multiple environmental and pathological stressful states and suppresses tumorigenesis. How AMPK protects the epithelium remained unknown until recently Aznar et al. identified GIV/Girdin as a novel effector of AMPK at the cell-cell junctions; phosphorylation of GIV at a single site by AMPK appears to be both necessary and sufficient for strengthening tight junctions and preserving cell polarity and epithelial barrier function in the face of energetic stress. Here we review the fundamentals of this specialized signaling pathway that buttresses cell-cell junctions against stress-induced collapse and discuss its pathophysiologic relevance in the context of a variety of diseases, including cancers, diabetes, aging, and the growing list of beneficial effects of the AMPK-activator, Metformin. PMID:28209925

Transforming Growth Factor β-1 Stimulates Profibrotic Epithelial Signaling to Activate Pericyte-Myofibroblast Transition in Obstructive Kidney Fibrosis

PubMed Central

Wu, Ching-Fang; Chiang, Wen-Chih; Lai, Chun-Fu; Chang, Fan-Chi; Chen, Yi-Ting; Chou, Yu-Hsiang; Wu, Ting-Hui; Linn, Geoffrey R.; Ling, Hong; Wu, Kwan-Dun; Tsai, Tun-Jun; Chen, Yung-Ming; Duffield, Jeremy S.; Lin, Shuei-Liong

2014-01-01

Pericytes have been identified as the major source of precursors of scar-producing myofibroblasts during kidney fibrosis. The underlying mechanisms triggering pericyte-myofibroblast transition are poorly understood. Transforming growth factor β-1 (TGF-β1) is well recognized as a pluripotent cytokine that drives organ fibrosis. We investigated the role of TGF-β1 in inducing profibrotic signaling from epithelial cells to activate pericyte-myofibroblast transition. Increased expression of TGF-β1 was detected predominantly in injured epithelium after unilateral ureteral obstruction, whereas downstream signaling from the TGF-β1 receptor increased in both injured epithelium and pericytes. In mice with ureteral obstruction that were treated with the pan anti–TGF-β antibody (1D11) or TGF-β receptor type I inhibitor (SB431542), kidney pericyte-myofibroblast transition was blunted. The consequence was marked attenuation of fibrosis. In addition, epithelial cell cycle G2/M arrest and production of profibrotic cytokines were both attenuated. Although TGF-β1 alone did not trigger pericyte proliferation in vitro, it robustly induced α smooth muscle actin (α-SMA). In cultured kidney epithelial cells, TGF-β1 stimulated G2/M arrest and production of profibrotic cytokines that had the capacity to stimulate proliferation and transition of pericytes to myofibroblasts. In conclusion, this study identified a novel link between injured epithelium and pericyte-myofibroblast transition through TGF-β1 during kidney fibrosis. PMID:23142380

Aqueous extracts and polysaccharides from Marshmallow roots (Althea officinalis L.): cellular internalisation and stimulation of cell physiology of human epithelial cells in vitro.

PubMed

Deters, Alexandra; Zippel, Janina; Hellenbrand, Nils; Pappai, Dirk; Possemeyer, Cathleen; Hensel, Andreas

2010-01-08

Aqueous extracts from the roots of Althea officinalis L. (Malvaceae) are widely used for treatment of irritated mucosa. The clinical proven effects are related to the presence of bioadhesive and mucilaginous polysaccharides from the rhamnogalacturonan type, leading to the physical formation of mucin-like on top of the irritated tissues. No data are available if the extracts or the polysaccharides from these extract exert an active influence on mucosal or connective tissue cells, in order to initiated changes in cell physiology, useful for better tissue regeneration. In vitro investigations of aqueous A. officinalis extract AE and raw polysaccharides (RPS) on epithelial KB cells and primary dermal human fibroblasts (pNHF) using WST1 vitality test and BrdU proliferation ELISA. Gene expression analysis by microarray from KB cells. Internalisation studies of polysaccharides were performed by laser scanning microscopy. AE (1, 10 microg/mL) had stimulating effect on cell viability and proliferation of epithelial KB cells. RPS (1, 10 microg/mL) stimulated cell vitality of epithelial cells significantly without triggering the cells into higher proliferation status. Neither AE nor RPS had any effect on fibroblasts. FITC-labeled RPS was shown to be internalised into epithelial cells, but not into fibroblasts. FITC-RPS was shown to form bioadhesive layers on the cell surface of dermal fibroblasts. Microarray analysis indicated an up-regulation of genes related to cell adhesion proteins, growth regulators, extracellular matrix, cytokine release and apoptosis. Aqueous extracts and polysaccharides from the roots of A. officinalis are effective stimulators of cell physiology of epithelial cells which can prove the traditional use of Marshmallow preparations for treatment of irritated mucous membranes within tissue regeneration. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

Bacillus thuringiensis toxins trigger receptor shedding from gypsy moth midgut cells

Treesearch

Algimantas P. Valaitis

2007-01-01

The mechanism of action of the Cry1 insecticidal proteins produced by Bacillus thuringiensis (Bt) begins with the processing of these proteins in the larval gut. After proteolytic activation, the Bt toxins bind to specific midgut receptors and insert into the membrane of the gut epithelial cells, causing insect death.

Mucin-mediated nanocarrier disassembly for triggered uptake of oligonucleotides as a delivery strategy for the potential treatment of mucosal tumours

NASA Astrophysics Data System (ADS)

Martirosyan, A.; Olesen, M. J.; Fenton, R. A.; Kjems, J.; Howard, K. A.

2016-06-01

This work demonstrates gastric mucin-triggered nanocarrier disassembly for release of antisense oligonucleotides and consequent unassisted cellular entry as a novel oral delivery strategy. A fluorescence activation-based reporter system was used to investigate the interaction and mucin-mediated disassembly of chitosan-based nanocarriers containing a 13-mer DNA oligonucleotide with a flanked locked RNA nucleic acid gapmer design. Gastric mucins were shown to trigger gapmer release from nanocarriers that was dependent on the interaction time, mucin concentration and N : P ratio with a maximal release at N : P 10. In contrast to siRNA, naked gapmers exhibited uptake into mucus producing HT-MTX mono-cultures and HT-MTX co-cultured with the carcinoma epithelial cell line Caco-2. Importantly, in vivo gapmer uptake was observed in epithelial tissue 30 min post-injection in murine intestinal loops. The findings present a mucosal design-based system tailored for local delivery of oligonucleotides that may maximize the effectiveness of gene silencing therapeutics within tumours at mucosal sites.This work demonstrates gastric mucin-triggered nanocarrier disassembly for release of antisense oligonucleotides and consequent unassisted cellular entry as a novel oral delivery strategy. A fluorescence activation-based reporter system was used to investigate the interaction and mucin-mediated disassembly of chitosan-based nanocarriers containing a 13-mer DNA oligonucleotide with a flanked locked RNA nucleic acid gapmer design. Gastric mucins were shown to trigger gapmer release from nanocarriers that was dependent on the interaction time, mucin concentration and N : P ratio with a maximal release at N : P 10. In contrast to siRNA, naked gapmers exhibited uptake into mucus producing HT-MTX mono-cultures and HT-MTX co-cultured with the carcinoma epithelial cell line Caco-2. Importantly, in vivo gapmer uptake was observed in epithelial tissue 30 min post-injection in murine intestinal loops. The findings present a mucosal design-based system tailored for local delivery of oligonucleotides that may maximize the effectiveness of gene silencing therapeutics within tumours at mucosal sites. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07206a

CD147 Promotes Entry of Pentamer-Expressing Human Cytomegalovirus into Epithelial and Endothelial Cells

PubMed Central

Pritchard, Sarah R.; Wisner, Todd W.; Liu, Jing; Jardetzky, Ted S.; Johnson, David C.

2018-01-01

ABSTRACT Human cytomegalovirus (HCMV) replicates in many diverse cell types in vivo, and entry into different cells involves distinct entry mechanisms and different envelope glycoproteins. HCMV glycoprotein gB is thought to act as the virus fusogen, apparently after being triggered by different gH/gL proteins that bind distinct cellular receptors or entry mediators. A trimer of gH/gL/gO is required for entry into all cell types, and entry into fibroblasts involves trimer binding to platelet-derived growth factor receptor alpha (PDGFRα). HCMV entry into biologically relevant epithelial and endothelial cells and monocyte-macrophages also requires a pentamer, gH/gL complexed with UL128, UL130, and UL131, and there is evidence that the pentamer binds unidentified receptors. We screened an epithelial cell cDNA library and identified the cell surface protein CD147, which increased entry of pentamer-expressing HCMV into HeLa cells but not entry of HCMV that lacked the pentamer. A panel of CD147-specific monoclonal antibodies inhibited HCMV entry into epithelial and endothelial cells, but not entry into fibroblasts. shRNA silencing of CD147 in endothelial cells inhibited HCMV entry but not entry into fibroblasts. CD147 colocalized with HCMV particles on cell surfaces and in endosomes. CD147 also promoted cell-cell fusion induced by expression of pentamer and gB in epithelial cells. However, soluble CD147 did not block HCMV entry and trimer and pentamer did not bind directly to CD147, supporting the hypothesis that CD147 acts indirectly through other proteins. CD147 represents the first HCMV entry mediator that specifically functions to promote entry of pentamer-expressing HCMV into epithelial and endothelial cells. PMID:29739904

TP53-dependent autophagy links the ATR-CHEK1 axis activation to proinflammatory VEGFA production in human bronchial epithelial cells exposed to fine particulate matter (PM2.5).

PubMed

Xu, Xiuduan; Wang, Hongli; Liu, Shasha; Xing, Chen; Liu, Yang; Aodengqimuge; Zhou, Wei; Yuan, Xiaoyan; Ma, Yongfu; Hu, Meiru; Hu, Yongliang; Zou, Shuxian; Gu, Ye; Peng, Shuangqing; Yuan, Shengtao; Li, Weiping; Ma, Yuanfang; Song, Lun

2016-10-02

ABSTARCT Epidemiological and clinical studies have increasingly shown that fine particulate matter (PM2.5) is associated with a number of pathological respiratory diseases, such as bronchitis, asthma, and chronic obstructive pulmonary disease, which share the common feature of airway inflammation induced by particle exposure. Thus, understanding how PM2.5 triggers inflammatory responses in the respiratory system is crucial for the study of PM2.5 toxicity. In the current study, we found that exposing human bronchial epithelial cells (immortalized Beas-2B cells and primary cells) to PM2.5 collected in the winter in Wuhan, a city in southern China, induced a significant upregulation of VEGFA (vascular endothelial growth factor A) production, a signaling event that typically functions to control chronic airway inflammation and vascular remodeling. Further investigations showed that macroautophagy/autophagy was induced upon PM2.5 exposure and then mediated VEGFA upregulation by activating the SRC (SRC proto-oncogene, non-receptor tyrosine kinase)-STAT3 (signal transducer and activator of transcription 3) pathway in bronchial epithelial cells. By exploring the upstream signaling events responsible for autophagy induction, we revealed a requirement for TP53 (tumor protein p53) activation and the expression of its downstream target DRAM1 (DNA damage regulated autophagy modulator 1) for the induction of autophagy. These results thus extend the role of TP53-DRAM1-dependent autophagy beyond cell fate determination under genotoxic stress and to the control of proinflammatory cytokine production. Moreover, PM2.5 exposure strongly induced the activation of the ATR (ATR serine/threonine kinase)-CHEK1/CHK1 (checkpoint kinase 1) axis, which subsequently triggered TP53-dependent autophagy and VEGFA production in Beas-2B cells. Therefore, these findings suggest a novel link between processes regulating genomic integrity and airway inflammation via autophagy induction in bronchial epithelial cells under PM2.5 exposure.

IκBNS induces Muc5ac expression in epithelial cells and causes airway hyper-responsiveness in murine asthma models.

PubMed

Yokota, M; Tamachi, T; Yokoyama, Y; Maezawa, Y; Takatori, H; Suto, A; Suzuki, K; Hirose, K; Takeda, K; Nakajima, H

2017-07-01

In allergic asthma, environmental allergens including house dust mite (HDM) trigger pattern recognition receptors and activate downstream signaling pathways including NF-κB pathways not only in immune cells but also in airway epithelial cells. Recent studies have shown that NF-κB activation is regulated positively or negatively depending on the cellular context by IκBNS (encoded by the gene Nfkbid), one of atypical IκB proteins, in the nucleus. Therefore, we hypothesized that IκBNS expressed in immune cells or epithelial cells is involved in the regulation of asthmatic responses. To determine the roles of IκBNS in HDM-induced asthmatic responses. Roles of IκBNS in HDM-induced airway inflammation and airway hyper-responsiveness (AHR) were examined by using IκBNS-deficient (Nfkbid -/- ) mice. Roles of IκBNS expressed in hematopoietic cells and nonhematopoietic cells were separately evaluated by bone marrow chimeric mice. Roles of IκBNS expressed in murine tracheal epithelial cells (mTECs) were examined by air-liquid interface culture. House dust mite-induced airway inflammation and AHR were exacerbated in mice lacking IκBNS in hematopoietic cells. In contrast, HDM-induced airway inflammation was exacerbated, but AHR was attenuated in mice lacking IκBNS in nonhematopoietic cells. The induction of Muc5ac, a representative mucin in asthmatic airways, was reduced in Nfkbid -/- mTEC, whereas the induction of Spdef, a master regulator of goblet cell metaplasia, was not impaired in Nfkbid -/- mTEC. Moreover, IκBNS bound to and activated the MUC5AC distal promoter in epithelial cells. IκBNS is involved in inducing Muc5ac expression in lung epithelial cells and causing AHR in HDM-induced asthma models. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Human intraepithelial lymphocytes.

PubMed

Mayassi, Toufic; Jabri, Bana

2018-04-20

The location of intraepithelial lymphocytes (IEL) between epithelial cells, their effector memory, cytolytic and inflammatory phenotype positions them to kill infected epithelial cells and protect the intestine against pathogens. Human TCRαβ + CD8αβ + IEL have the dual capacity to recognize modified self via natural killer (NK) receptors (autoreactivity) as well as foreign antigen via the T cell receptor (TCR), which is accomplished in mouse by two cell subsets, the naturally occurring TCRαβ + CD8αα + and adaptively induced TCRαβ + CD8αβ + IEL subsets, respectively. The private/oligoclonal nature of the TCR repertoire of both human and mouse IEL suggests local environmental factors dictate the specificity of IEL responses. The line between sensing of foreign antigens and autoreactivity is blurred for IEL in celiac disease, where recognition of stress ligands by induced activating NK receptors in conjunction with inflammatory signals such as IL-15 can result in low-affinity TCR/non-cognate antigen and NK receptor/stress ligand interactions triggering destruction of intestinal epithelial cells.

SUPPRESSION OF THE EPITHELIAL-MESENCHYMAL TRANSITION BY GRAINYHEAD-LIKE-2

PubMed Central

Cieply, Benjamin; Riley, Philip; Pifer, Phillip M.; Widmeyer, Joseph; Addison, Joseph B.; Ivanov, Alexey V.; Denvir, James; Frisch, Steven M.

2012-01-01

Grainyhead genes are involved in wound healing and developmental neural tube closure. In light of the high degree of similarity between the epithelial-mesenchymal transitions (EMT) occurring in wound healing processes and the cancer stem cell-like compartment of tumors, including TGF-β-dependence, we investigated the role of the Grainyhead gene, Grainyhead-Like-2 (GRHL2) in oncogenic EMT. GRHL2 was down-regulated specifically in the claudin-low subclass breast tumors and in basal-B subclass breast cancer cell lines. GRHL2 suppressed TGF-β-induced, Twist-induced or spontaneous EMT, enhanced anoikis-sensitivity, and suppressed mammosphere generation in mammary epithelial cells. These effects were mediated in part by suppression of ZEB1 expression via direct repression of the ZEB1 promoter. GRHL2 also inhibited Smad-mediated transcription and it upregulated mir200b/c as well as the TGF-β receptor antagonist, BMP2. Lastly, ectopic expression of GRHL2 in MDA-MB-231 breast cancer cells triggered a mesenchymal-to-epithelial transition and restored sensitivity to anoikis. Taken together, our findings define a major role for GRHL2 in the suppression of oncogenic EMT in breast cancer cells. PMID:22379025

A newly established bovine intestinal epithelial cell line is effective for in vitro screening of potential antiviral immunobiotic microorganisms for cattle.

PubMed

Chiba, Eriko; Villena, Julio; Hosoya, Shoichi; Takanashi, Naoya; Shimazu, Tomoyuki; Aso, Hisashi; Tohno, Masanori; Suda, Yoshihito; Kawai, Yasushi; Saito, Tadao; Miyazawa, Kenji; He, Fang; Kitazawa, Haruki

2012-10-01

We evaluated whether a bovine intestinal epithelial (BIE) cell line could serve as a useful in vitro model system for studying antiviral immune responses in bovine intestinal epithelial cells (IECs) and for the primary screening of immunobiotic microorganisms with antiviral protective capabilities. Immunofluorescent analyses revealed that toll-like receptor 3 (TLR3) was expressed in BIE cells, and the results of real-time quantitative PCR showed that these cells respond to stimulation with poly(I:C) by up-regulating pro-inflammatory cytokines and type I interferons. In addition, we demonstrated that BIE cells are useful for the primary screening of immunobiotic lactic acid bacteria strains which are able to beneficially modulate antiviral immune responses triggered by TLR3 activation in bovine IECs. The characterization of BIE cells performed in the present study represents an important step towards the establishment of a valuable bovine in vitro system that could be used for the development of immunomodulatory feed for bovine hosts. Copyright © 2011 Elsevier Ltd. All rights reserved.

Th-POK regulates mammary gland lactation through mTOR-SREBP pathway.

PubMed

Zhang, Rui; Ma, Huimin; Gao, Yuan; Wu, Yanjun; Qiao, Yuemei; Geng, Ajun; Cai, Cheguo; Han, Yingying; Zeng, Yi Arial; Liu, Xiaolong; Ge, Gaoxiang

2018-02-01

The Th-inducing POK (Th-POK, also known as ZBTB7B or cKrox) transcription factor is a key regulator of lineage commitment of immature T cell precursors. It is yet unclear the physiological functions of Th-POK besides helper T cell differentiation. Here we show that Th-POK is restrictedly expressed in the luminal epithelial cells in the mammary glands that is upregulated at late pregnancy and lactation. Lineage restrictedly expressed Th-POK exerts distinct biological functions in the mammary epithelial cells and T cells in a tissue-specific manner. Th-POK is not required for mammary epithelial cell fate determination. Mammary gland morphogenesis in puberty and alveologenesis in pregnancy are phenotypically normal in the Th-POK-deficient mice. However, Th-POK-deficient mice are defective in triggering the onset of lactation upon parturition with large cellular lipid droplets retained within alveolar epithelial cells. As a result, Th-POK knockout mice are unable to efficiently secret milk lipid and to nurse the offspring. Such defect is mainly attributed to the malfunctioned mammary epithelial cells, but not the tissue microenvironment in the Th-POK deficient mice. Th-POK directly regulates expression of insulin receptor substrate-1 (IRS-1) and insulin-induced Akt-mTOR-SREBP signaling. Th-POK deficiency compromises IRS-1 expression and Akt-mTOR-SREBP signaling in the lactating mammary glands. Conversely, insulin induces Th-POK expression. Thus, Th-POK functions as an important feed-forward regulator of insulin signaling in mammary gland lactation.

Th-POK regulates mammary gland lactation through mTOR-SREBP pathway

PubMed Central

Wu, Yanjun; Qiao, Yuemei; Geng, Ajun; Cai, Cheguo; Han, Yingying; Zeng, Yi Arial

2018-01-01

The Th-inducing POK (Th-POK, also known as ZBTB7B or cKrox) transcription factor is a key regulator of lineage commitment of immature T cell precursors. It is yet unclear the physiological functions of Th-POK besides helper T cell differentiation. Here we show that Th-POK is restrictedly expressed in the luminal epithelial cells in the mammary glands that is upregulated at late pregnancy and lactation. Lineage restrictedly expressed Th-POK exerts distinct biological functions in the mammary epithelial cells and T cells in a tissue-specific manner. Th-POK is not required for mammary epithelial cell fate determination. Mammary gland morphogenesis in puberty and alveologenesis in pregnancy are phenotypically normal in the Th-POK-deficient mice. However, Th-POK-deficient mice are defective in triggering the onset of lactation upon parturition with large cellular lipid droplets retained within alveolar epithelial cells. As a result, Th-POK knockout mice are unable to efficiently secret milk lipid and to nurse the offspring. Such defect is mainly attributed to the malfunctioned mammary epithelial cells, but not the tissue microenvironment in the Th-POK deficient mice. Th-POK directly regulates expression of insulin receptor substrate-1 (IRS-1) and insulin-induced Akt-mTOR-SREBP signaling. Th-POK deficiency compromises IRS-1 expression and Akt-mTOR-SREBP signaling in the lactating mammary glands. Conversely, insulin induces Th-POK expression. Thus, Th-POK functions as an important feed-forward regulator of insulin signaling in mammary gland lactation. PMID:29420538

BDE-99 (2,2',4,4',5-pentabromodiphenyl ether) triggers epithelial-mesenchymal transition in colorectal cancer cells via PI3K/Akt/Snail signaling pathway.

PubMed

Wang, Fei; Ruan, Xin-Jian; Zhang, Hong-Yan

2015-01-01

The gut is in direct contact with BDE-99 (2,2',4,4',5-pentabromodiphenyl ether), one of the most abundant PBDE congeners in the environment and in human tissues. The objective of the present study was to investigate the effects of BDE-99 on colorectal cancer (CRC) cells. The effects of BDE-99 on cell proliferation were measured by CCK-8 assay in the CRC cell line HCT-116. Wound healing and transwell migration/invasion assays were used to test the migration and invasion of CRC cells. Factors related to epithelial-to-mesenchymal transition (EMT) were measured by real-time PCR and Western blot analysis for mRNA and protein levels, respectively. BDE-99 was found to increase migration and invasion and trigger EMT in HCT-116 cells; EMT was characterized by cells acquiring mesenchymal spindle-like morphology and by increased expression of N-cadherin with a concomitant decrease in E-cadherin. BDE-99 treatment also increased the protein and mRNA levels of the transcription factor Snail, but not Slug, Twist, and ZEB1. Knockdown of Snail by siRNA significantly attenuated BDE-99-induced EMT in HCT-116 cells, suggesting that Snail plays a crucial role in BDE-99-induced EMT. The PI3K/Akt inhibitor LY294002 completely blocked BDE-99-induced Snail and invasion of HCT-116 cells. Our results revealed that BDE-99 can trigger the EMT of colon cancer cells via the PI3K/AKT/Snail signaling pathway. This study provides new insight into the tumorigenesis and metastasis of CRC stimulated by BDE-99 and possibly other PBDE congeners.

Hypo-responsiveness of interleukin-8 production in human embryonic epithelial intestine 407 cells independent of NF-{kappa}B pathway: New lessons from endotoxin and ribotoxic deoxynivalenol

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

Moon, Yuseok; Yang, Hyun; Park, Seung-Hwan

Mucosal epithelium senses external toxic insults and transmits the danger signals into the epithelial cells in order to activate a broad range of inflammatory responses. However, pre-exposure to the commensal endotoxins can induce inflammatory tolerance and maintain the homeostasis without excessive immune responses. We recently reported that ribotoxin deoxynivalenol (DON) and its derivatives elicited the pro-inflammatory response as the mucosal insults in human epithelial cells. Taking the knowledge into consideration, we tested the hypothesis that endotoxin pre-exposure can attenuate ribotoxin-induced epithelial interleukin-8 (IL-8) production via a tolerance mechanism. Pre-exposure to endotoxin repressed IL-8 release and its gene expression. However, inflammatorymore » tolerance was not mediated by the attenuated NF-{kappa}B activation which has been generally recognized as the major mediator of LPS-mediated toll-like receptor (TLR) signaling pathway. Instead, pre-exposure to endotoxin was observed to trigger the delayed induction of peroxisome proliferator-activated receptor gamma (PPAR-{gamma}) which contributed to the diminished IL-8 production in the human epithelial cells. Moreover, endogenous PPAR-{gamma} agonist suppressed toxicant-mediated interleukin-8 production and IL-8 mRNA stability. Taken together, endotoxin induced hypo-production of pro-inflammatory cytokine IL-8 in the human epithelial cells, which was associated with the delayed activation of PPAR-{gamma} expression by pre-existing endotoxin.« less

Mesothelioma: Identification of the Key Molecular Events Triggered by BAP1

DTIC Science & Technology

2016-04-01

with 5ml of phosphate-buffered saline. The peritoneal cells obtained were pelleted and supernatant was removed for later cytokine analysis. Cells were...Interestingly, BAP1 has been recently shown to regulate the myeloid stem cell compartment via complex alterations of the transcriptional profile, possibly via...regulation of IL-6 activation by asbestos in lung epithelial cells : role of reactive oxygen species. J Immunol 1997; 159: 3921–3928. 43 Occupational

Reactive oxygen species induced by Streptococcus pyogenes invasion trigger apoptotic cell death in infected epithelial cells.

PubMed

Aikawa, Chihiro; Nozawa, Takashi; Maruyama, Fumito; Tsumoto, Kohei; Hamada, Shigeyuki; Nakagawa, Ichiro

2010-06-01

Streptococcus pyogenes (group A streptococcus, GAS), one of the most common pathogens of humans, attaches and invades into human pharyngeal or skin epithelial cells. We have previously reported that induction of apoptosis is associated with GAS invasion, which induces mitochondrial dysfunction and apoptotic cell death. We demonstrate here that GAS-induced apoptosis is mediated by reactive oxygen species (ROS) production. Both the induction of apoptosis and ROS production markedly increased upon invasion of wild-type GAS strain JRS4 into HeLa cells; however, the apoptotic response was not observed in fibronectin-binding protein F1-disrupted mutant SAM1-infected cells. In Bcl-2-overexpressing HeLa cells (HBD98-2-4), the induction of apoptosis, ROS production and mitochondrial dysfunction were significantly suppressed, whereas the numbers of invaded GAS was not different between HeLa (mock cells) and the HeLa HBD98-2-4 cells. Whereas Rac1 activation occurred during GAS invasion, ROS production in GAS-infected cells was clearly inhibited by transfection with the Rac1 mutants (L37 or V12L37), but not by the dominant active mutant (V12L61) or by the dominant negative mutant (N17). These observations indicate that GAS invasion triggers ROS production through Rac1 activation and generated ROS induced mitochondrial dysfunction leading to cellular apoptosis.

Dual modes of motility at the leading edge of migrating epithelial cell sheets

PubMed Central

Klarlund, Jes K.

2012-01-01

Purse-string healing is driven by contraction of actin/myosin cables that span cells at wound edges, and it is the predominant mode of closing small round wounds in embryonic and some adult epithelia. Wounds can also heal by cell crawling, and my colleagues and I have shown previously that the presence of unconstrained, straight edges in sheets of epithelial cells is a sufficient signal to induce healing by crawling. Here, it is reported that the presence of highly concave edges, which are free or physically constrained by an inert material (agarose), is sufficient to induce formation of purse strings. It was determined that neither of the two types of healing required cell damage or other potential stimuli by using the particularly gentle procedure of introducing gaps by digesting agarose blocks imbedded in the cell sheets. Movement by crawling depends on signaling by the EGF receptor (EGFR); however, this was not required for purse-string contraction. A migrating epithelial cell sheet usually produces finger-like projections of crawling cells. The cells between fingers contain continuous actin cables, which were also determined to contain myosin IIA and exhibit additional characteristics of purse strings. When crawling was blocked by inhibition of EGFR signaling, the concave regions continued to move, suggesting that both mechanisms contribute to propel the sheets forward. Wounding epithelial cell sheets causes activation of the EGFR, which triggers movement by crawling. The EGFR was found to be activated only at straight and convex edges, which explains how both types of movement can coexist at leading epithelial edges. PMID:23019364

A role for the substance P/NK-1 receptor complex in cell proliferation and apoptosis in oral lichen planus.

PubMed

González Moles, M A; Esteban, F; Ruiz-Avila, I; Gil Montoya, J A; Brener, S; Bascones-Martínez, A; Muñoz, M

2009-03-01

To determine whether substance P (SP) and NK-1 receptor (NK-1R) are expressed in oral lichen planus (OLP) and are related to cell proliferation and apoptosis in this disease. Tissue samples from 50 OLP patients and 26 healthy controls were studied. Immunohistochemistry was performed with anti-SP, anti-NK-1R, anti-ki-67 and anti-caspase-3 monoclonal antibodies and the clinical and pathological data of the OLP patients were evaluated. With the exception of NK-1R expression in epithelial cell membrane and cytoplasm, all markers were more frequently present in OLP patients than in controls (P < 0.05). Higher cytoplasmatic expression of NK-1R was associated with higher epithelial expression of caspase-3 (P < 0.05). Higher epithelial expression of NK-1R and SP was associated with higher suprabasal and basal epithelial expression of ki-67 (P < 0.05 and P < 0.005, respectively). Actions of the SP/NK-1R complex may contribute to the immune disorder underlying OLP and trigger stimuli to induce cell proliferation. These results indicate that this complex might play a role in the malignant transformation of OLP.

The environmental carcinogen benzo[a]pyrene induces a Warburg-like metabolic reprogramming dependent on NHE1 and associated with cell survival

PubMed Central

Hardonnière, Kévin; Saunier, Elise; Lemarié, Anthony; Fernier, Morgane; Gallais, Isabelle; Héliès-Toussaint, Cécile; Mograbi, Baharia; Antonio, Samantha; Bénit, Paule; Rustin, Pierre; Janin, Maxime; Habarou, Florence; Ottolenghi, Chris; Lavault, Marie-Thérèse; Benelli, Chantal; Sergent, Odile; Huc, Laurence; Bortoli, Sylvie; Lagadic-Gossmann, Dominique

2016-01-01

Cancer cells display alterations in many cellular processes. One core hallmark of cancer is the Warburg effect which is a glycolytic reprogramming that allows cells to survive and proliferate. Although the contributions of environmental contaminants to cancer development are widely accepted, the underlying mechanisms have to be clarified. Benzo[a]pyrene (B[a]P), the prototype of polycyclic aromatic hydrocarbons, exhibits genotoxic and carcinogenic effects, and it is a human carcinogen according to the International Agency for Research on Cancer. In addition to triggering apoptotic signals, B[a]P may induce survival signals, both of which are likely to be involved in cancer promotion. We previously suggested that B[a]P-induced mitochondrial dysfunctions, especially membrane hyperpolarization, might trigger cell survival signaling in rat hepatic epithelial F258 cells. Here, we further characterized these dysfunctions by focusing on energy metabolism. We found that B[a]P promoted a metabolic reprogramming. Cell respiration decreased and lactate production increased. These changes were associated with alterations in the tricarboxylic acid cycle which likely involve a dysfunction of the mitochondrial complex II. The glycolytic shift relied on activation of the Na+/H+ exchanger 1 (NHE1) and appeared to be a key feature in B[a]P-induced cell survival related to changes in cell phenotype (epithelial-to-mesenchymal transition and cell migration). PMID:27488617

A novel role for integrin-linked kinase in epithelial sheet morphogenesis.

PubMed

Vespa, Alisa; D'Souza, Sudhir J A; Dagnino, Lina

2005-09-01

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca(2+) triggers formation of cell-cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca(2+) treatment of keratinocytes induces rapid (6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell-cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/beta-catenin to cell borders, precluding Ca(2+)-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell-cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell-cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration.

Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells

NASA Astrophysics Data System (ADS)

Han, Meng; Blindewald-Wittich, Almut; Holz, Frank G.; Giese, Günter; Niemz, Markolf H.; Snyder, Sarah; Sun, Hui; Yu, Jiayi; Agopov, Michael; La Schiazza, Olivier; Bille, Josef F.

2006-01-01

Degeneration of retinal pigment epithelial (RPE) cells severely impairs the visual function of retina photoreceptors. However, little is known about the events that trigger the death of RPE cells at the subcellular level. Two-photon excited autofluorescence (TPEF) imaging of RPE cells proves to be well suited to investigate both the morphological and the spectral characteristics of the human RPE cells. The dominant fluorophores of autofluorescence derive from lipofuscin (LF) granules that accumulate in the cytoplasm of the RPE cells with increasing age. Spectral TPEF imaging reveals the existence of abnormal LF granules with blue shifted autofluorescence in RPE cells of aging patients and brings new insights into the complicated composition of the LF granules. Based on a proposed two-photon laser scanning ophthalmoscope, TPEF imaging of the living retina may be valuable for diagnostic and pathological studies of age related eye diseases.

CD44S-hyaluronan interactions protect cells resulting from EMT against anoikis

PubMed Central

Cieply, Benjamin; Koontz, Colton; Frisch, Steven M.

2016-01-01

The detachment of normal epithelial cells from matrix triggers an apoptotic response known as anoikis, during homeostatic turnover. Metastatic tumor cells evade anoikis, by mechanisms that are only partly characterized. In particular, the epithelial–mesenchymal transition (EMT) in a subset of invasive tumor cells confers anoikis-resistance. In some cases, EMT up-regulates the cancer stem cell marker CD44S and the enzyme hyaluronic acid synthase-2 (HAS2). CD44S is the major receptor for hyaluronan in the extracellular matrix. Herein, we demonstrate that CD44S, unlike the CD44E isoform expressed in normal epithelial cells, contributes to the protection against anoikis. This protection requires the interaction of CD44S with hyaluronan (HA). CD44S–HA interaction is proposed to play an important role in tumor metastasis through enhanced cell survival under detached conditions. PMID:25937513

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

PubMed

Bonnet, Mariette; Tran Van Nhieu, Guy

2016-01-01

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

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

PubMed Central

Bonnet, Mariette; Tran Van Nhieu, Guy

2016-01-01

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

Dysregulated Estrogen Receptor Signaling in the Hypothalamic-Pituitary-Ovarian Axis Leads to Ovarian Epithelial Tumorigenesis in Mice

PubMed Central

Laws, Mary J.; Kannan, Athilakshmi; Pawar, Sandeep; Haschek, Wanda M.; Bagchi, Milan K.; Bagchi, Indrani C.

2014-01-01

The etiology of ovarian epithelial cancer is poorly understood, mainly due to the lack of an appropriate experimental model for studying the onset and progression of this disease. We have created a mutant mouse model in which aberrant estrogen receptor alpha (ERα) signaling in the hypothalamic-pituitary-ovarian axis leads to ovarian epithelial tumorigenesis. In these mice, termed ERαd/d, the ERα gene was conditionally deleted in the anterior pituitary, but remained intact in the hypothalamus and the ovary. The loss of negative-feedback regulation by estrogen (E) at the level of the pituitary led to increased production of luteinizing hormone (LH) by this tissue. Hyperstimulation of the ovarian cells by LH resulted in elevated steroidogenesis, producing high circulating levels of steroid hormones, including E. The ERαd/d mice exhibited formation of palpable ovarian epithelial tumors starting at 5 months of age with 100% penetrance. By 15 months of age, 80% of ERαd/d mice die. Besides proliferating epithelial cells, these tumors also contained an expanded population of luteinized stromal cells, which acquire the ability to express P450 aromatase and synthesize E locally. In response to the elevated levels of E, the ERα signaling was accentuated in the ovarian epithelial cells of ERαd/d mice, triggering increased ERα-dependent gene expression, abnormal cell proliferation, and tumorigenesis. Consistent with these findings, treatment of ERαd/d mice with letrozole, an aromatase inhibitor, markedly reduced circulating E and ovarian tumor volume. We have, therefore, developed a unique animal model, which serves as a useful tool for exploring the involvement of E-dependent signaling pathways in ovarian epithelial tumorigenesis. PMID:24603706

Hepatocyte growth factor activator inhibitors (HAI-1 and HAI-2): Emerging key players in epithelial integrity and cancer.

PubMed

Kataoka, Hiroaki; Kawaguchi, Makiko; Fukushima, Tsuyoshi; Shimomura, Takeshi

2018-03-01

The growth, survival, and metabolic activities of multicellular organisms at the cellular level are regulated by intracellular signaling, systemic homeostasis and the pericellular microenvironment. Pericellular proteolysis has a crucial role in processing bioactive molecules in the microenvironment and thereby has profound effects on cellular functions. Hepatocyte growth factor activator inhibitor type 1 (HAI-1) and HAI-2 are type I transmembrane serine protease inhibitors expressed by most epithelial cells. They regulate the pericellular activities of circulating hepatocyte growth factor activator and cellular type II transmembrane serine proteases (TTSPs), proteases required for the activation of hepatocyte growth factor (HGF)/scatter factor (SF). Activated HGF/SF transduces pleiotropic signals through its receptor tyrosine kinase, MET (coded by the proto-oncogene MET), which are necessary for cellular migration, survival, growth and triggering stem cells for accelerated healing. HAI-1 and HAI-2 are also required for normal epithelial functions through regulation of TTSP-mediated activation of other proteases and protease-activated receptor 2, and also through suppressing excess degradation of epithelial junctional proteins. This review summarizes current knowledge regarding the mechanism of pericellular HGF/SF activation and highlights emerging roles of HAIs in epithelial development and integrity, as well as tumorigenesis and progression of transformed epithelial cells. © 2018 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.

A Novel Role for Integrin-linked Kinase in Epithelial Sheet Morphogenesis

PubMed Central

Vespa, Alisa; D'Souza, Sudhir J.A.; Dagnino, Lina

2005-01-01

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca2+ triggers formation of cell–cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca2+ treatment of keratinocytes induces rapid (≤1 h) translocation to the cell membrane of the adherens junction (AJ) proteins E-cadherin and β-catenin. This is followed by slower (>6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell–cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/β-catenin to cell borders, precluding Ca2+-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell–cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell–cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration. PMID:15975904

Chemosensors in the Nose: Guardians of the Airways

PubMed Central

Tizzano, Marco

2013-01-01

The G-protein-coupled receptor molecules and downstream effectors that are used by taste buds to detect sweet, bitter, and savory tastes are also utilized by chemoresponsive cells of the airways to detect irritants. Here, we describe the different cell types in the airways that utilize taste-receptor signaling to trigger protective epithelial and neural responses to potentially dangerous toxins and bacterial infection. PMID:23280357

Lactobacillus delbrueckii subsp lactis CIDCA 133 modulates response of human epithelial and dendritic cells infected with Bacillus cereus.

PubMed

Rolny, I S; Tiscornia, I; Racedo, S M; Pérez, P F; Bollati-Fogolín, M

2016-11-30

It is known that probiotic microorganisms are able to modulate pathogen virulence. This ability is strain dependent and involves multiple interactions between microorganisms and relevant host's cell populations. In the present work we focus on the effect of a potentially probiotic lactobacillus strain (Lactobacillus delbrueckii subsp. lactis CIDCA 133) in an in vitro model of Bacillus cereus infection. Our results showed that infection of intestinal epithelial HT-29 cells by B. cereus induces nuclear factor kappa B (NF-κB) pathway. Noteworthy, the presence of strain L. delbrueckii subsp.lactis CIDCA 133 increases stimulation. However, B. cereus-induced interleukin (IL)-8 production by epithelial cells is partially abrogated by L. delbrueckii subsp. lactis CIDCA 133. These findings suggest that signalling pathways other than that of NF-κB are involved. In a co-culture system (HT-29 and monocyte-derived dendritic cells), B. cereus was able to translocate from the epithelial (upper) to the dendritic cell compartment (lower). This translocation was partially abrogated by the presence of lactobacilli in the upper compartment. In addition, infection of epithelial cells in the co-culture model, led to an increase in the expression of CD86 by dendritic cells. This effect could not be modified in the presence of lactobacilli. Interestingly, infection of enterocytes with B. cereus triggers production of proinflammatory cytokines by dendritic cells (IL-8, IL-6 and tumour necrosis factor alpha (TNF-α)). The production of TNF-α (a protective cytokine in B. cereus infections) by dendritic cells was increased in the presence of lactobacilli. The present work demonstrates for the first time the effect of L. delbrueckii subsp. lactis CIDCA 133, a potentially probiotic strain, in an in vitro model of B. cereus infection. The presence of the probiotic strain modulates cell response both in infected epithelial and dendritic cells thus suggesting a possible beneficial effect of selected lactobacilli strains on the course of B. cereus infection.

Type 2 innate lymphoid cells disrupt bronchial epithelial barrier integrity by targeting tight junctions through IL-13 in asthmatic patients.

PubMed

Sugita, Kazunari; Steer, Catherine A; Martinez-Gonzalez, Itziar; Altunbulakli, Can; Morita, Hideaki; Castro-Giner, Francesc; Kubo, Terufumi; Wawrzyniak, Paulina; Rückert, Beate; Sudo, Katsuko; Nakae, Susumu; Matsumoto, Kenji; O'Mahony, Liam; Akdis, Mübeccel; Takei, Fumio; Akdis, Cezmi A

2018-01-01

Bronchial epithelial barrier leakiness and type 2 innate lymphoid cells (ILC2s) have been separately linked to asthma pathogenesis; however, the influence of ILC2s on the bronchial epithelial barrier has not been investigated previously. We investigated the role of ILC2s in the regulation of bronchial epithelial tight junctions (TJs) and barrier function both in bronchial epithelial cells of asthmatic patients and healthy subjects and general innate lymphoid cell- and ILC2-deficient mice. Cocultures of human ILC2s and bronchial epithelial cells were used to determine transepithelial electrical resistance, paracellular flux, and TJ mRNA and protein expressions. The effect of ILC2s on TJs was examined by using a murine model of IL-33-induced airway inflammation in wild-type, recombination-activating gene 2 (Rag2) -/- , Rag2 -/- Il2rg -/- , and Rora sg/sg mice undergoing bone marrow transplantation to analyze the in vivo relevance of barrier disruption by ILC2s. ILC2s significantly impaired the epithelial barrier, as demonstrated by reduced transepithelial electrical resistance and increased fluorescein isothiocyanate-dextran permeability in air-liquid interface cultures of human bronchial epithelial cells. This was in parallel to decreased mRNAs and disrupted protein expression of TJ proteins and was restored by neutralization of IL-13. Intranasal administration of recombinant IL-33 to wild-type and Rag2 -/- mice lacking T and B cells triggered TJ disruption, whereas Rag2 -/- Il2rg -/- and Rora sg/sg mice undergoing bone marrow transplantation that lack ILC2s did not show any barrier leakiness. Direct nasal administration of IL-13 was sufficient to induce deficiency in the TJ barrier in the bronchial epithelium of mice in vivo. These data highlight an essential mechanism in asthma pathogenesis by demonstrating that ILC2s are responsible for bronchial epithelial TJ barrier leakiness through IL-13. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Autophagy regulates tissue overgrowth in a context-dependent manner.

PubMed

Pérez, E; Das, G; Bergmann, A; Baehrecke, E H

2015-06-01

Autophagy is a catabolic process that has been implicated both as a tumor suppressor and in tumor progression. Here, we investigate this dichotomy in cancer biology by studying the influence of altered autophagy in Drosophila models of tissue overgrowth. We find that the impact of altered autophagy depends on both genotype and cell type. As previously observed in mammals, decreased autophagy suppresses Ras-induced eye epithelial overgrowth. In contrast, autophagy restricts epithelial overgrowth in a Notch-dependent eye model. Even though decreased autophagy did not influence Hippo pathway-triggered overgrowth, activation of autophagy strongly suppresses this eye epithelial overgrowth. Surprisingly, activation of autophagy enhanced Hippo pathway-driven overgrowth in glia cells. These results indicate that autophagy has different influences on tissue growth in distinct contexts, and highlight the importance of understanding the influence of autophagy on growth to augment a rationale therapeutic strategy.

FAK Regulates Intestinal Epithelial Cell Survival and Proliferation during Mucosal Wound Healing

PubMed Central

Tilghman, Robert W.; Casanova, James E.; Bouton, Amy H.

2011-01-01

Background Following damage to the intestinal epithelium, restoration of epithelial barrier integrity is triggered by a robust proliferative response. In other tissues, focal adhesion kinase (FAK) regulates many of the cellular processes that are critical for epithelial homeostasis and restitution, including cell migration, proliferation and survival. However, few studies to date have determined how FAK contributes to mucosal wound healing in vivo. Methodology and Principal Findings To examine the role of FAK in intestinal epithelial homeostasis and during injury, we generated intestinal epithelium (IE)-specific conditional FAK knockout mice. Colitis was induced with dextran-sulfate-sodium (DSS) and intestinal tissues were analyzed by immunohistochemistry and immunoblotting. While intestinal development occurred normally in mice lacking FAK, FAK-deficient animals were profoundly susceptible to colitis. The loss of epithelial FAK resulted in elevated p53 expression and an increased sensitivity to apoptosis, coincident with a failure to upregulate epithelial cell proliferation. FAK has been reported to function as a mechanosensor, inducing cyclin D1 expression and promoting cell cycle progression under conditions in which tissue/matrix stiffness is increased. Collagen deposition, a hallmark of inflammatory injury resulting in increased tissue rigidity, was observed in control and FAK knockout mice during colitis. Despite this fibrotic response, the colonic epithelium in FAK-deficient mice exhibited significantly reduced cyclin D1 expression, suggesting that proliferation is uncoupled from fibrosis in the absence of FAK. In support of this hypothesis, proliferation of Caco-2 cells increased proportionally with matrix stiffness in vitro only under conditions of normal FAK expression; FAK depleted cells exhibited reduced proliferation concomitant with attenuated cyclin D1 expression. Conclusions In the colon, FAK functions as a regulator of epithelial cell survival and proliferation under conditions of mucosal injury and a mechanosensor of tissue compliance, inducing repair-driven proliferation in the colonic epithelium through upregulation of cyclin D1. PMID:21887232

Long term exposure to environmental concentrations of diesel exhaust particles does not impact the phenotype of human bronchial epithelial cells.

PubMed

Savary, Camille C; Bellamri, Nessrine; Morzadec, Claudie; Langouët, Sophie; Lecureur, Valérie; Vernhet, Laurent

2018-06-19

Chronic exposure to diesel engine exhausts is associated with an increased risk of pulmonary diseases including lung cancer. Diesel engine exhausts contain large amounts of diesel exhaust particles (DEP) on which are adsorbed several carcinogenic compounds such as polycyclic aromatic hydrocarbons. Acute toxicity of high concentrations of DEP has been largely demonstrated in various in vitro cellular models. In contrast, the cellular and molecular impacts of low environmental concentrations of DEP on the phenotype of chronically exposed lung epithelial cells remain to be investigated. In the present study, we show that long term exposure (6 months) to 2 μg/ml (0.4 μg/cm 2 ) DEP (standard reference material 1650b) increased cytochrome P4501A mRNA levels in the human bronchial epithelial BEAS-2B cell line. However, chronic exposure to DEP did not change cell morphology, trigger epithelial-mesenchymal transition or increase anchorage-independent cell growth. Moreover, DEP increase neither the levels of reactive oxygen species or those of γ-histone H2AX, nor the expression of interleukin-6 and interleukin-8. Our results thus demonstrate that the chronic exposure to low DEP concentrations could increase cytochrome P501A gene expression in BEAS-2B cells but did not induce molecular effects related to genotoxicity, oxidative stress or inflammation. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

PubMed

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

2013-08-27

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

CD147/EMMPRIN acts as a functional entry receptor for measles virus on epithelial cells.

PubMed

Watanabe, Akira; Yoneda, Misako; Ikeda, Fusako; Terao-Muto, Yuri; Sato, Hiroki; Kai, Chieko

2010-05-01

Measles is a highly contagious human disease caused by measles virus (MeV) and remains the leading cause of death in children, particularly in developing countries. Wild-type MeV preferentially infects lymphocytes by using signaling lymphocytic activation molecule (SLAM), whose expression is restricted to hematopoietic cells, as a receptor. MeV also infects other epithelial and neuronal cells that do not express SLAM and causes pneumonia and diarrhea and, sometimes, serious symptoms such as measles encephalitis and subacute sclerosing panencephalitis. The discrepancy between the tissue tropism of MeV and the distribution of SLAM-positive cells suggests that there are unknown receptors other than SLAM for MeV. Here we identified CD147/EMMPRIN (extracellular matrix metalloproteinase inducer), a transmembrane glycoprotein, which acts as a receptor for MeV on epithelial cells. Furthermore, we found the incorporation of cyclophilin B (CypB), a cellular ligand for CD147, in MeV virions, and showed that inhibition of CypB incorporation significantly attenuated SLAM-independent infection on epithelial cells, while it had no effect on SLAM-dependent infection. To date, MeV infection was considered to be triggered by binding of its hemagglutinin (H) protein and cellular receptors. Our present study, however, indicates that MeV infection also occurs via CD147 and virion-associated CypB, independently of MeV H. Since CD147 is expressed in a variety of cells, including epithelial and neuronal cells, this molecule possibly functions as an entry receptor for MeV in SLAM-negative cells. This is the first report among members of the Mononegavirales that CD147 is used as a virus entry receptor via incorporated CypB in the virions.

[Etiopathogenic aspects in development and evolution of pterigyum].

PubMed

Crăiţoiu, Stefania; Ciprian, Livezeanu; Rodica, Mănescu; Mihai, Afrem; Anca, Eremia Irina

2008-01-01

Pterygium is an epithelial hyperplasia accompanied by a fibrovascular growth originating at the corneo-conjunctival junction, from where the modified limbic cells migrate and surpass the cornea. The studies reviewed show that it is an active process associated with cell growth, remodelling of the connective tissue, angiogenesis and inflammation. Despite the lack of knowledge regarding the pathogenesis of pterygia, epidemiologic evidence suggests that exposure to UV-irradiation may be an initial trigger in the development of this lesion. Other theories include changes of the apoptotic pathway the presence of some active angiogenetic factors or involvement of the MMPs, cytokines and growths factors. UV light could be the initial trigger that activates epithelial cells at or near the limbus to produce cytokines such as IL-6 and IL-8 and growth factors. These multifactorial proteins set up a cascade of events that include inflammation, proliferation, angiogenesis and antiapoptosis. Cytokines are able to induce the expression of MMPs and their tissue inhibitors (TIMPs) making it likely that they would also affect the rate of tissue remodeling, such as destruction of Bowman's membrane and the invasion of pterygium. In the etiology of pterygium abnormalities in tear functions have also been emphasized.

Low-Dose Alkylphenol Exposure Promotes Mammary Epithelium Alterations and Transgenerational Developmental Defects, But Does Not Enhance Tumorigenic Behavior of Breast Cancer Cells

PubMed Central

Chamard-Jovenin, Clémence; Thiebaut, Charlène; Chesnel, Amand; Bresso, Emmanuel; Morel, Chloé; Smail-Tabbone, Malika; Devignes, Marie-Dominique; Boukhobza, Taha; Dumond, Hélène

2017-01-01

Fetal and neonatal exposure to long-chain alkylphenols has been suspected to promote breast developmental disorders and consequently to increase breast cancer risk. However, disease predisposition from developmental exposures remains unclear. In this work, human MCF-10A mammary epithelial cells were exposed in vitro to a low dose of a realistic (4-nonylphenol + 4-tert-octylphenol) mixture. Transcriptome and cell-phenotype analyses combined to functional and signaling network modeling indicated that long-chain alkylphenols triggered enhanced proliferation, migration ability, and apoptosis resistance and shed light on the underlying molecular mechanisms which involved the human estrogen receptor alpha 36 (ERα36) variant. A male mouse-inherited transgenerational model of exposure to three environmentally relevant doses of the alkylphenol mix was set up in order to determine whether and how it would impact on mammary gland architecture. Mammary glands from F3 progeny obtained after intrabuccal chronic exposure of C57BL/6J P0 pregnant mice followed by F1–F3 male inheritance displayed an altered histology which correlated with the phenotypes observed in vitro in human mammary epithelial cells. Since cellular phenotypes are similar in vivo and in vitro and involve the unique ERα36 human variant, such consequences of alkylphenol exposure could be extrapolated from mouse model to human. However, transient alkylphenol treatments combined to ERα36 overexpression in mammary epithelial cells were not sufficient to trigger tumorigenesis in xenografted Nude mice. Therefore, it remains to be determined if low-dose alkylphenol transgenerational exposure and subsequent abnormal mammary gland development could account for an increased breast cancer susceptibility. PMID:29109696

Typhoid fever as a triggering factor in acute and intractable bronchial asthma attack.

PubMed

Wardhana; Surachmanto, Eko E; Datau, E A

2013-10-01

Typhoid fever is an enteric infection caused by Salmonella typhi. In Indonesia, typhoid fever is endemic with high incidence of the disease. In daily practice we frequently have patients with bronchial asthma, and it is becoming worse when these patients get typhoid fever. After oral ingestion, Salmonella typhi invades the the intestine mucosa after conducted by microbial binding to epithelial cells, destroying the microfold cells (M cell) then passed through the lamina propria and detected by dendritic cells (DC) which express a variety of pathogen recognition receptors on the surfaces, including Toll-Like Receptor (TLR). expressed on macrophages and on intestinal epithelial cells inducing degradation of IB, and translocation of NF-B (Nuclear Factor-Kappa Beta). This process initiates the induction of pro-inflammatory gene expression profile adhesion molecules, chemokines, adhesion molecules, and other proteins that induce and perpetuate the inflammation in host cells then will induce acute ant intractable attack of bronchial asthma. The role of typhoid fever in bronchial asthma, especially in persons with acute attack of bronchial asthma, is not well understood. In this article, we will discuss the role of typhoid fever in the bronchial asthma patients which may cause bronchial asthma significantly become more severe even triggering the acute and intractable attack of bronchial asthma. This fact makes an important point, to treat completely the typhoid fever in patients with bronchial asthma.

Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2.

PubMed

Li, Qiong; Kumar, Ashok; Gui, Jian-Fang; Yu, Fu-Shin X

2008-05-01

Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-kappaB, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-kappaB activation in HUCL cells after lipoprotein lipase treatment and in cell lines expressing TLR4 or TLR9, but not TLR2, indicating lipoprotein nature of the extracts. saLP induced the up-regulation of a variety of inflammatory cytokines and chemokines (IL-6, IL-8, ICAM-1), antimicrobial molecules (hBD-2, LL-37, and iNOS), and homeostasis genes (Mn-SOD) at both the mRNA level and protein level. Similar inflammatory response to saLP was also observed in primarily cultured HCECs using the production of IL-6 as readout. Moreover, TLR2 neutralizing antibody blocked the saLP-induced secretion of IL-6, IL-8 and hBD2 in HUCL cells. Our findings suggest that saLP activates TLR2 and triggers innate immune response in the cornea to S. aureus infection via production of proinflammatory cytokines and defense molecules.

Advanced Oxidation Protein Products Induce Epithelial-Mesenchymal Transition of Intestinal Epithelial Cells via a PKC δ-Mediated, Redox-Dependent Signaling Pathway.

PubMed

Xu, Xiaoping; Sun, Shibo; Xie, Fang; Ma, Juanjuan; Tang, Jing; He, Shuying; Bai, Lan

2017-07-01

Epithelial-mesenchymal transition (EMT) has been considered a fundamental mechanism in complications of Crohn's disease (CD), especially intestinal fibrosis. However, the mechanism underlying EMT regulation in intestinal fibrosis remains unclear. This study aimed to investigate the role of advanced oxidation protein products (AOPPs) in the occurrence of intestinal EMT. AOPPs accumulated in CD tissues and were associated with EMT marker expression in fibrotic lesions from CD patients. Challenge with AOPPs induced intestinal epithelial cell (IEC) phenotype transdifferentiation, fibroblast-like phenotype acquisition, and production of extracellular matrix, both in vitro and in vivo. The effect of AOPPs was mainly mediated by a protein kinase C (PKC) δ-mediated redox-dependent pathway, including phosphorylation of PKC δ, recruitment of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, production of reactive oxygen species, and NF-κB p65 activation. Inhibition of AOPP-redox signaling activation effectively blocked AOPP-induced EMT in vitro. Studies performed in normal rats showed that chronic administration of AOPPs triggered the occurrence of EMT in rat intestinal epithelia, accompanied by disruption of intestinal integrity, and by promotion of collagen deposition. These effects could be reversed by inhibition of NADPH oxidase. Innovation and Conclusion: This is the first study to demonstrate that AOPPs triggered the occurrence of EMT in IECs in vitro and in vivo through PKC δ-mediated redox-dependent signaling. Our study identifies the role of AOPPs and, in turn, EMT in intestinal fibrosis and provides novel potential targets for the treatment of intestinal fibrotic diseases. Antioxid. Redox Signal. 27, 37-56.

CD147/EMMPRIN Acts as a Functional Entry Receptor for Measles Virus on Epithelial Cells▿

PubMed Central

Watanabe, Akira; Yoneda, Misako; Ikeda, Fusako; Terao-Muto, Yuri; Sato, Hiroki; Kai, Chieko

2010-01-01

Measles is a highly contagious human disease caused by measles virus (MeV) and remains the leading cause of death in children, particularly in developing countries. Wild-type MeV preferentially infects lymphocytes by using signaling lymphocytic activation molecule (SLAM), whose expression is restricted to hematopoietic cells, as a receptor. MeV also infects other epithelial and neuronal cells that do not express SLAM and causes pneumonia and diarrhea and, sometimes, serious symptoms such as measles encephalitis and subacute sclerosing panencephalitis. The discrepancy between the tissue tropism of MeV and the distribution of SLAM-positive cells suggests that there are unknown receptors other than SLAM for MeV. Here we identified CD147/EMMPRIN (extracellular matrix metalloproteinase inducer), a transmembrane glycoprotein, which acts as a receptor for MeV on epithelial cells. Furthermore, we found the incorporation of cyclophilin B (CypB), a cellular ligand for CD147, in MeV virions, and showed that inhibition of CypB incorporation significantly attenuated SLAM-independent infection on epithelial cells, while it had no effect on SLAM-dependent infection. To date, MeV infection was considered to be triggered by binding of its hemagglutinin (H) protein and cellular receptors. Our present study, however, indicates that MeV infection also occurs via CD147 and virion-associated CypB, independently of MeV H. Since CD147 is expressed in a variety of cells, including epithelial and neuronal cells, this molecule possibly functions as an entry receptor for MeV in SLAM-negative cells. This is the first report among members of the Mononegavirales that CD147 is used as a virus entry receptor via incorporated CypB in the virions. PMID:20147391

Uropathogenic E. coli Exploit CEA to Promote Colonization of the Urogenital Tract Mucosa

PubMed Central

Muenzner, Petra; Kengmo Tchoupa, Arnaud; Klauser, Benedikt; Brunner, Thomas; Putze, Johannes; Dobrindt, Ulrich; Hauck, Christof R.

2016-01-01

Attachment to the host mucosa is a key step in bacterial pathogenesis. On the apical surface of epithelial cells, members of the human carcinoembryonic antigen (CEA) family are abundant glycoproteins involved in cell-cell adhesion and modulation of cell signaling. Interestingly, several gram-negative bacterial pathogens target these receptors by specialized adhesins. The prototype of a CEACAM-binding pathogen, Neisseria gonorrhoeae, utilizes colony opacity associated (Opa) proteins to engage CEA, as well as the CEA-related cell adhesion molecules CEACAM1 and CEACAM6 on human epithelial cells. By heterologous expression of neisserial Opa proteins in non-pathogenic E. coli we find that the Opa protein-CEA interaction is sufficient to alter gene expression, to increase integrin activity and to promote matrix adhesion of infected cervical carcinoma cells and immortalized vaginal epithelial cells in vitro. These CEA-triggered events translate in suppression of exfoliation and improved colonization of the urogenital tract by Opa protein-expressing E. coli in CEA-transgenic compared to wildtype mice. Interestingly, uropathogenic E. coli expressing an unrelated CEACAM-binding protein of the Afa/Dr adhesin family recapitulate the in vitro and in vivo phenotype. In contrast, an isogenic strain lacking the CEACAM-binding adhesin shows reduced colonization and does not suppress epithelial exfoliation. These results demonstrate that engagement of human CEACAMs by distinct bacterial adhesins is sufficient to blunt exfoliation and to promote host infection. Our findings provide novel insight into mucosal colonization by a common UPEC pathotype and help to explain why human CEACAMs are a preferred epithelial target structure for diverse gram-negative bacteria to establish a foothold on the human mucosa. PMID:27171273

ROLE OF RAS IN METAL-INDUCED EGF RECEPTOR AND NFKB SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

EPA Science Inventory

We have shown previously that EGF receptor signaling is triggered by some metals associated with ambient air particles. Western blot using phospho-specific antibodies showed that As, Zn and V activated EGF receptor tyrosine kinase and the downstream kinases, MEK1/2 and ERK1/2. Us...

Structural centrosome aberrations promote non-cell-autonomous invasiveness.

PubMed

Ganier, Olivier; Schnerch, Dominik; Oertle, Philipp; Lim, Roderick Yh; Plodinec, Marija; Nigg, Erich A

2018-05-02

Centrosomes are the main microtubule-organizing centers of animal cells. Although centrosome aberrations are common in tumors, their consequences remain subject to debate. Here, we studied the impact of structural centrosome aberrations, induced by deregulated expression of ninein-like protein (NLP), on epithelial spheres grown in Matrigel matrices. We demonstrate that NLP-induced structural centrosome aberrations trigger the escape ("budding") of living cells from epithelia. Remarkably, all cells disseminating into the matrix were undergoing mitosis. This invasive behavior reflects a novel mechanism that depends on the acquisition of two distinct properties. First, NLP-induced centrosome aberrations trigger a re-organization of the cytoskeleton, which stabilizes microtubules and weakens E-cadherin junctions during mitosis. Second, atomic force microscopy reveals that cells harboring these centrosome aberrations display increased stiffness. As a consequence, mitotic cells are pushed out of mosaic epithelia, particularly if they lack centrosome aberrations. We conclude that centrosome aberrations can trigger cell dissemination through a novel, non-cell-autonomous mechanism, raising the prospect that centrosome aberrations contribute to the dissemination of metastatic cells harboring normal centrosomes. © 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

Airway inflammation in cystic fibrosis: molecular mechanisms and clinical implications.

PubMed

Cohen-Cymberknoh, Malena; Kerem, Eitan; Ferkol, Thomas; Elizur, Arnon

2013-12-01

Airway epithelial cells and immune cells participate in the inflammatory process responsible for much of the pathology found in the lung of patients with cystic fibrosis (CF). Intense bronchial neutrophilic inflammation and release of proteases and oxygen radicals perpetuate the vicious cycle and progressively damage the airways. In vitro studies suggest that CF transmembrane conductance regulator (CFTR)-deficient airway epithelial cells display signalling abnormalities and aberrant intracellular processes which lead to transcription of inflammatory mediators. Several transcription factors, especially nuclear factor-κB, are activated. In addition, the accumulation of abnormally processed CFTR in the endoplasmic reticulum results in unfolded protein responses that trigger 'cell stress' and apoptosis leading to dysregulation of the epithelial cells and innate immune function in the lung, resulting in exaggerated and ineffective airway inflammation. Measuring airway inflammation is crucial for initiating treatment and monitoring its effect. No inflammatory biomarker predictive for the clinical course of CF lung disease is currently known, although neutrophil elastase seems to correlate with lung function decline. CF animal models mimicking human lung disease may provide an important insight into the pathogenesis of lung inflammation in CF and identify new therapeutic targets.

Systematic Analysis of Cell-Type Differences in the Epithelial Secretome Reveals Insights into the Pathogenesis of RSV-Induced Lower Respiratory Tract Infections

PubMed Central

Zhao, Yingxin; Jamaluddin, Mohammad; Zhang, Yueqing; Sun, Hong; Ivanciuc, Teodora; Garofalo, Roberto P.; Brasier, Allan R.

2017-01-01

Lower respiratory tract infections (LRTIs) from Respiratory Syncytial Virus (RSV) are due, in part, to secreted signals from lower airway cells that modify immune response and trigger airway remodeling. To understand this process, we applied an unbiased quantitative proteomics analysis of the RSV-induced epithelial secretory response in cells representative of the trachea (hBECs) vs small airway bronchiolar cells (hSAECs). A workflow was established using telomerase- immortalized human epithelial cells that revealed highly reproducible cell type-specific differences in both secreted proteins and nanoparticles (exosomes). Approximately one-third of secretome proteins are exosomal, with the remainder from lysosomal and vacuolar compartments. We applied this workflow to three independently derived primary human cultures from trachea (phBECs) vs bronchioles (phSAECs). 577 differentially expressed proteins from control supernatants and 966 differentially expressed proteins from RSV-infected cell supernatants were identified at a 1% false discovery rate (FDR). Fifteen proteins unique to RSV-infected phBECs were regulated by epithelial-specific ets homology factor (EHF). 106 proteins unique to RSV-infected hSAECs were regulated by the transcription factor NFκB. In this latter group, we validated the differential expression of Chemokine (C-C Motif) Ligand 20 (CCL20)/macrophage-inducible protein (MIP)3α, thymic stromal lymphopoietin (TSLP) and chemokine (CC) ligand 3-like 1(CCL3-L1) because of their roles in Th2 polarization. CCL20/MIP3α was the most active mucin-inducing factor in the RSV-infected hSAEC secretome, and was differentially expressed in smaller airways in a mouse model of RSV infection. These studies provide insights into the complexity of innate responses, and regional differences in epithelial secretome participating in RSV LRTI-induced airway remodeling. PMID:28258195

The Effect of K+ on Caspase Activity of Corneal Epithelial Cells Exposed to UVB

PubMed Central

Leerar, John R.; Glupker, Courtney D.; Schotanus, Mark P.; Ubels, John L.

2016-01-01

Exposure of human corneal limbal epithelial (HCLE) cells to UVB triggers rapid loss of K+ and apoptosis via activation of caspases-9, -8 and -3. It has been shown that preventing loss of intracellular K+ can inhibit apoptosis. The goal of this study was to investigate the effect of K+ on the UVB-induced caspase activity. HCLE cells were exposed to 150 mJ/cm2 UVB, followed by measurement of caspase activity in cell lysates. Caspase activity was measured in the presence and absence of 100 mM K+ in the reaction buffer. UVB-induced activity of caspases-9, -8 and -3 all decreased in the presence of 100 mM K+. These results suggest that a role of high [K+] in the cell is to inhibit caspase activity. Therefore, when cells lose K+ in response to UVB, caspases are activated and cells go into apoptosis. This supports our hypothesis that K+ inhibits caspase activity. PMID:27456133

Mitosis can drive cell cannibalism through entosis

PubMed Central

Durgan, Joanne; Tseng, Yun-Yu; Hamann, Jens C; Domart, Marie-Charlotte; Collinson, Lucy; Overholtzer, Michael; Florey, Oliver

2017-01-01

Entosis is a form of epithelial cell cannibalism that is prevalent in human cancer, typically triggered by loss of matrix adhesion. Here, we report an alternative mechanism for entosis in human epithelial cells, driven by mitosis. Mitotic entosis is regulated by Cdc42, which controls mitotic morphology. Cdc42 depletion enhances mitotic deadhesion and rounding, and these biophysical changes, which depend on RhoA activation and are phenocopied by Rap1 inhibition, permit subsequent entosis. Mitotic entosis occurs constitutively in some human cancer cell lines and mitotic index correlates with cell cannibalism in primary human breast tumours. Adherent, wild-type cells can act efficiently as entotic hosts, suggesting that normal epithelia may engulf and kill aberrantly dividing neighbours. Finally, we report that Paclitaxel/taxol promotes mitotic rounding and subsequent entosis, revealing an unconventional activity of this drug. Together, our data uncover an intriguing link between cell division and cannibalism, of significance to both cancer and chemotherapy. DOI: http://dx.doi.org/10.7554/eLife.27134.001 PMID:28693721

Adenovirus E4ORF1-induced MYC activation promotes host cell anabolic glucose metabolism and virus replication

PubMed Central

Thai, Minh; Graham, Nicholas A; Braas, Daniel; Nehil, Michael; Komisopoulou, Evangelia; Kurdistani, Siavash K.; McCormick, Frank; Graeber, Thomas G.; Christofk, Heather R.

2014-01-01

SUMMARY Virus infections trigger metabolic changes in host cells that support the bioenergetic and biosynthetic demands of viral replication. While recent studies have characterized virus-induced changes in host cell metabolism (Munger et al., 2008; Terry et al., 2012), the molecular mechanisms by which viruses reprogram cellular metabolism have remained elusive. Here we show that the gene product of adenovirus E4ORF1 is necessary for adenovirus-induced upregulation of host cell glucose metabolism and sufficient to promote enhanced glycolysis in cultured epithelial cells by activation of MYC. E4ORF1 localizes to the nucleus, binds to MYC, and enhances MYC binding to glycolytic target genes, resulting in elevated expression of specific glycolytic enzymes. E4ORF1 activation of MYC promotes increased nucleotide biosynthesis from glucose intermediates and enables optimal adenovirus replication in primary lung epithelial cells. Our findings show how a viral protein exploits host cell machinery to reprogram cellular metabolism and promote optimal progeny virion generation. PMID:24703700

Epstein-Barr virus glycoprotein gB and gHgL can mediate fusion and entry in trans, and heat can act as a partial surrogate for gHgL and trigger a conformational change in gB.

PubMed

Chesnokova, Liudmila S; Ahuja, Munish K; Hutt-Fletcher, Lindsey M

2014-11-01

Epstein-Barr virus (EBV) fusion with an epithelial cell requires virus glycoproteins gHgL and gB and is triggered by an interaction between gHgL and integrin αvβ5, αvβ6, or αvβ8. Fusion with a B cell requires gHgL, gp42, and gB and is triggered by an interaction between gp42 and human leukocyte antigen class II. We report here that, like alpha- and betaherpesviruses, EBV, a gammaherpesvirus, can mediate cell fusion if gB and gHgL are expressed in trans. Entry of a gH-null virus into an epithelial cell is possible if the epithelial cell expresses gHgL, and entry of the same virus, which phenotypically lacks gHgL and gp42, into a B cell expressing gHgL is possible in the presence of a soluble integrin. Heat is capable of inducing the fusion of cells expressing only gB, and the proteolytic digestion pattern of gB in virions changes in the same way following the exposure of virus to heat or to soluble integrins. It is suggested that the Gibbs free energy released as a result of the high-affinity interaction of gHgL with an integrin contributes to the activation energy required to cause the refolding of gB from a prefusion to a postfusion conformation. The core fusion machinery of herpesviruses consists of glycoproteins gB and gHgL. We demonstrate that as in alpha- and betaherpesvirus, gB and gHgL of the gammaherpesvirus EBV can mediate fusion and entry when expressed in trans in opposing membranes, implicating interactions between the ectodomains of the proteins in the activation of fusion. We further show that heat and exposure to a soluble integrin, both of which activate fusion, result in the same changes in the proteolytic digestion pattern of gB, possibly representing the refolding of gB from its prefusion to its postfusion conformation. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Epstein-Barr Virus Glycoprotein gB and gHgL Can Mediate Fusion and Entry in trans, and Heat Can Act as a Partial Surrogate for gHgL and Trigger a Conformational Change in gB

PubMed Central

Chesnokova, Liudmila S.; Ahuja, Munish K.

2014-01-01

ABSTRACT Epstein-Barr virus (EBV) fusion with an epithelial cell requires virus glycoproteins gHgL and gB and is triggered by an interaction between gHgL and integrin αvβ5, αvβ6, or αvβ8. Fusion with a B cell requires gHgL, gp42, and gB and is triggered by an interaction between gp42 and human leukocyte antigen class II. We report here that, like alpha- and betaherpesviruses, EBV, a gammaherpesvirus, can mediate cell fusion if gB and gHgL are expressed in trans. Entry of a gH-null virus into an epithelial cell is possible if the epithelial cell expresses gHgL, and entry of the same virus, which phenotypically lacks gHgL and gp42, into a B cell expressing gHgL is possible in the presence of a soluble integrin. Heat is capable of inducing the fusion of cells expressing only gB, and the proteolytic digestion pattern of gB in virions changes in the same way following the exposure of virus to heat or to soluble integrins. It is suggested that the Gibbs free energy released as a result of the high-affinity interaction of gHgL with an integrin contributes to the activation energy required to cause the refolding of gB from a prefusion to a postfusion conformation. IMPORTANCE The core fusion machinery of herpesviruses consists of glycoproteins gB and gHgL. We demonstrate that as in alpha- and betaherpesvirus, gB and gHgL of the gammaherpesvirus EBV can mediate fusion and entry when expressed in trans in opposing membranes, implicating interactions between the ectodomains of the proteins in the activation of fusion. We further show that heat and exposure to a soluble integrin, both of which activate fusion, result in the same changes in the proteolytic digestion pattern of gB, possibly representing the refolding of gB from its prefusion to its postfusion conformation. PMID:25142593

IFN-γ Induces Mimic Extracellular Trap Cell Death in Lung Epithelial Cells Through Autophagy-Regulated DNA Damage.

PubMed

Lin, Chiou-Feng; Chien, Shun-Yi; Chen, Chia-Ling; Hsieh, Chia-Yuan; Tseng, Po-Chun; Wang, Yu-Chih

2016-02-01

Treatment of interferon-γ (IFN-γ) causes cell growth inhibition and cytotoxicity in lung epithelial malignancies. Regarding the induction of autophagy related to IFN-γ signaling, this study investigated the link between autophagy and IFN-γ cytotoxicity. In A549 human lung cancer cells, IFN-γ treatment induced concurrent apoptotic and nonapoptotic events. Unexpectedly, the nonapoptotic cells present mimic extracellular trap cell death (ETosis), which was regulated by caspase-3 and by autophagy induction through immunity-related GTPase family M protein 1 and activating transcription factor 6. Furthermore, IFN-γ signaling controlled mimic ETosis through a mechanism involving an autophagy- and Fas-associated protein with death domain-controlled caspase-8/-3 activation. Following caspase-mediated lamin degradation, IFN-γ caused DNA damage-associated ataxia telangiectasia and Rad3-related protein (ATR)/ataxia telangiectasia mutated (ATM)-regulated mimic ETosis. Upon ATR/ATM signaling, peptidyl arginine deiminase 4 (PAD4)-mediated histone 3 citrullination promoted mimic ETosis. Such IFN-γ-induced effects were defective in PC14PE6/AS2 human lung cancer cells, which were unsusceptible to IFN-γ-induced autophagy. Due to autophagy-based caspase cascade activation, IFN-γ triggers unconventional caspase-mediated DNA damage, followed by ATR/ATM-regulated PAD4-mediated histone citrullination during mimic ETosis in lung epithelial malignancy.

Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish

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

Chenais, Nathalie; Lareyre, Jean-Jacques; Le Bail, Pierre-Yves

The development of fin primary cell cultures for in vitro cellular and physiological studies is hampered by slow cell outgrowth, low proliferation rate, poor viability, and sparse cell characterization. Here, we investigated whether the recycling of fresh explants after a first conventional culture could improve physiological stability and sustainability of the culture. The recycled explants were able to give a supplementary cell culture showing faster outgrowth, cleaner cell layers and higher net cell production. The cells exhibited a highly stabilized profile for marker gene expression including a low cytokeratin 49 (epithelial marker) and a high collagen 1a1 (mesenchymal marker) expression.more » Added to the cell spindle-shaped morphology, motility behavior, and actin organization, this suggests that the cells bore stable mesenchymal characteristics. This contrast with the time-evolving expression pattern observed in the control fresh explants during the first 2 weeks of culture: a sharp decrease in cytokeratin 49 expression was concomitant with a gradual increase in col1a1. We surmise that such loss of epithelial features for the benefit of mesenchymal ones was triggered by an epithelial to mesenchymal transition (EMT) process or by way of a progressive population replacement process. Overall, our findings provide a comprehensive characterization of this new primary culture model bearing mesenchymal features and whose stability over culture time makes those cells good candidates for cell reprogramming prior to nuclear transfer, in a context of fish genome preservation. - Highlights: • Recycled fin explants outgrow cells bearing stable mesenchymal traits. • Cell production and quality is enhanced in the recycled explant culture system. • Fresh fin primary culture is highly variable and loose epithelial traits over time.« less

cAMP enhances Cx43 gap junction formation and function and reverses choline deficiency apoptosis.

PubMed

Albright, C D; Kuo, J; Jeong, S

2001-08-01

Previously, it had been shown that acute choline deficiency (CD) induced apoptosis in cultured rat liver epithelial cells, whereas cells that are adapted to survive in low-choline-containing medium acquire resistance to CD apoptosis and undergo malignant transformation. Thus, understanding the mechanisms of action of CD could increase our understanding of the role of choline, an essential nutrient, in the process of malignant transformation. The present experiments were designed to test the hypothesis that CD might function as a pro-apoptotic trigger by altering the localization of connexin 43 gap junction protein and gap junctional intercellular communication (GJIC). Established liver epithelial cells (WB cells; Hep3B cells) were maintained in a defined, serum-free medium control (70 microM choline) or choline deficient medium (CD, 5 microM choline) and the localization of connexin 43 protein (Cx43) was studied by immunocytochemistry and Western blotting. In nontumorigenic WB cells, CD apoptosis was associated with retention of Cx43 in the golgi/ER region of the cytoplasm and decreased GJIC as measured using a preloading fluorescent dye transfer method (calcein AM/DiIC(18)). Cells maintained in CD in the presence of 8-bromoadenosine 3':5'-cyclic monophosphate exhibited restoration of Cx43 at the plasma membrane and increased GJIC and inhibition of apoptosis. These studies show that CD apoptosis in nontumorigenic liver epithelial cells is associated with alterations to Cx43 and GJIC and that an uncoupling of Cx43 localization and GJIC is related to resistance to CD apoptosis in transformed liver epithelial cells. Copyright 2001 Academic Press.

Pathophysiology of ocular surface squamous neoplasia

PubMed Central

Gichuhi, Stephen; Ohnuma, Shin-ichi; Sagoo, Mandeep S.; Burton, Matthew J.

2014-01-01

The incidence of ocular surface squamous neoplasia (OSSN) is strongly associated with solar ultraviolet (UV) radiation, HIV and human papilloma virus (HPV). Africa has the highest incidence rates in the world. Most lesions occur at the limbus within the interpalpebral fissure particularly the nasal sector. The nasal limbus receives the highest intensity of sunlight. Limbal epithelial crypts are concentrated nasally and contain niches of limbal epithelial stem cells in the basal layer. It is possible that these are the progenitor cells in OSSN. OSSN arises in the basal epithelial cells spreading towards the surface which resembles the movement of corneo-limbal stem cell progeny before it later invades through the basement membrane below. UV radiation damages DNA producing pyrimidine dimers in the DNA chain. Specific CC → TT base pair dimer transformations of the p53 tumour-suppressor gene occur in OSSN allowing cells with damaged DNA past the G1-S cell cycle checkpoint. UV radiation also causes local and systemic photoimmunosuppression and reactivates latent viruses such as HPV. The E7 proteins of HPV promote proliferation of infected epithelial cells via the retinoblastoma gene while E6 proteins prevent the p53 tumour suppressor gene from effecting cell-cycle arrest of DNA-damaged and infected cells. Immunosuppression from UV radiation, HIV and vitamin A deficiency impairs tumour immune surveillance allowing survival of aberrant cells. Tumour growth and metastases are enhanced by; telomerase reactivation which increases the number of cell divisions a cell can undergo; vascular endothelial growth factor for angiogenesis and matrix metalloproteinases (MMPs) that destroy the intercellular matrix between cells. Despite these potential triggers, the disease is usually unilateral. It is unclear how HPV reaches the conjunctiva. PMID:25447808

Establishment and characterization of a telomerase immortalized human gingival epithelial cell line.

PubMed

Moffatt-Jauregui, C E; Robinson, B; de Moya, A V; Brockman, R D; Roman, A V; Cash, M N; Culp, D J; Lamont, R J

2013-12-01

Gingival keratinocytes are used in model systems to investigate the interaction between periodontal bacteria and the epithelium in the initial stages of the periodontal disease process. Primary gingival epithelial cells (GECs) have a finite lifespan in culture before they enter senescence and cease to replicate, while epithelial cells immortalized with viral proteins can exhibit chromosomal rearrangements. The aim of this study was to generate a telomerase immortalized human gingival epithelial cell line and compare its in vitro behaviour to that of human GECs. Human primary GECs were immortalized with a bmi1/hTERT combination to prevent cell cycle triggers of senescence and telomere shortening. The resultant cell-line, telomerase immortalized gingival keratinocytes (TIGKs), were compared to GECs for cell morphology, karyotype, growth and cytokeratin expression, and further characterized for replicative lifespan, expression of toll-like receptors and invasion by P. gingivalis. TIGKs showed morphologies, karyotype, proliferation rates and expression of characteristic cytokeratin proteins comparable to GECs. TIGKs underwent 36 passages without signs of senescence and expressed transcripts for toll-like receptors 1-6, 8 and 9. A subpopulation of cells underwent stratification after extended time in culture. The cytokeratin profiles of TIGK monolayers were consistent with basal cells. When allowed to stratify, cytokeratin profiles of TIGKs were consistent with suprabasal cells of the junctional epithelium. Further, TIGKs were comparable to GECs in previously reported levels and kinetics of invasion by wild-type P. gingivalis and an invasion defective ΔserB mutant. Results confirm bmi1/hTERT immortalization of primary GECs generated a robust cell line with similar characteristics to the parental cell type. TIGKs represent a valuable model system for the study of oral bacteria interactions with host gingival cells. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Multimerization of Adenovirus Serotype 3 Fiber Knob Domains Is Required for Efficient Binding of Virus to Desmoglein 2 and Subsequent Opening of Epithelial Junctions▿

PubMed Central

Wang, Hongjie; Li, ZongYi; Yumul, Roma; Lara, Stephanie; Hemminki, Akseli; Fender, Pascal; Lieber, André

2011-01-01

Recently, we identified desmoglein 2 (DSG2) as the main receptor for a group of species B adenoviruses (Ads), including Ad3, a serotype that is widely distributed in the human population (H. Wang et al., Nat. Med. 17:96–104, 2011). In this study, we have attempted to delineate structural details of the Ad3 interaction with DSG2. For CAR- and CD46-interacting Ad serotypes, attachment to cells can be completely blocked by an excess of recombinant fiber knob protein, while soluble Ad3 fiber knob only inefficiently blocks Ad3 infection. We found that the DSG2-interacting domain(s) within Ad3 is formed by several fiber knob domains that have to be in the spatial constellation that is present in viral particles. Based on this finding, we generated a small recombinant, self-dimerizing protein containing the Ad3 fiber knob (Ad3-K/S/Kn). Ad3-K/S/Kn bound to DSG2 with high affinity and blocked Ad3 infection. We demonstrated by confocal immunofluorescence and transmission electron microscopy analyses that Ad3-K/S/Kn, through its binding to DSG2, triggered the transient opening of intercellular junctions in epithelial cells. The pretreatment of epithelial cells with Ad3-K/S/Kn resulted in increased access to receptors that are localized in or masked by epithelial junctions, e.g., CAR or Her2/neu. Ad3-K/S/Kn treatment released CAR from tight junctions and thus increased the transduction of epithelial cells by a serotype Ad5-based vector. Furthermore, the pretreatment of Her2/neu-positive breast cancer cells with Ad3-K/S/Kn increased the killing of cancer cells by the Her2/neu-targeting monoclonal antibody trastuzumab (Herceptin). This study widens our understanding of how Ads achieve high avidity to their receptors and the infection of epithelial tissue. The small recombinant protein Ad3-K/S/Kn has practical implications for the therapy of epithelial cancer and gene/drug delivery to normal epithelial tissues. PMID:21525338

Transcriptional Profiling of Ectoderm Specification to Keratinocyte Fate in Human Embryonic Stem Cells

PubMed Central

Tadeu, Ana Mafalda Baptista; Lin, Samantha; Hou, Lin; Chung, Lisa; Zhong, Mei; Zhao, Hongyu; Horsley, Valerie

2015-01-01

In recent years, several studies have shed light into the processes that regulate epidermal specification and homeostasis. We previously showed that a broad-spectrum γ–secretase inhibitor DAPT promoted early keratinocyte specification in human embryonic stem cells triggered to undergo ectoderm specification. Here, we show that DAPT accelerates human embryonic stem cell differentiation and induces expression of the ectoderm protein AP2. Furthermore, we utilize RNA sequencing to identify several candidate regulators of ectoderm specification including those involved in epithelial and epidermal development in human embryonic stem cells. Genes associated with transcriptional regulation and growth factor activity are significantly enriched upon DAPT treatment during specification of human embryonic stem cells to the ectoderm lineage. The human ectoderm cell signature identified in this study contains several genes expressed in ectodermal and epithelial tissues. Importantly, these genes are also associated with skin disorders and ectodermal defects, providing a platform for understanding the biology of human epidermal keratinocyte development under diseased and homeostatic conditions. PMID:25849374

Chemo-mechanical modeling of tumor growth in elastic epithelial tissue

NASA Astrophysics Data System (ADS)

Bratsun, Dmitry A.; Zakharov, Andrey P.; Pismen, Len

2016-08-01

We propose a multiscale chemo-mechanical model of the cancer tumor development in the epithelial tissue. The epithelium is represented by an elastic 2D array of polygonal cells with its own gene regulation dynamics. The model allows the simulation of the evolution of multiple cells interacting via the chemical signaling or mechanically induced strain. The algorithm includes the division and intercalation of cells as well as the transformation of normal cells into a cancerous state triggered by a local failure of the spatial synchronization of the cellular rhythms driven by transcription/translation processes. Both deterministic and stochastic descriptions of the system are given for chemical signaling. The transformation of cells means the modification of their respective parameters responsible for chemo-mechanical interactions. The simulations reproduce a distinct behavior of invasive and localized carcinoma. Generally, the model is designed in such a way that it can be readily modified to take account of any newly understood gene regulation processes and feedback mechanisms affecting chemo-mechanical properties of cells.

Barrier-protective function of intestinal epithelial Toll-like receptor 2.

PubMed

Cario, E

2008-11-01

The intestinal epithelial cell (IEC) barrier plays an important role in maintaining mucosal immune homeostasis. Dysregulated IEC barrier function appears to trigger and perpetuate inflammation in inflammatory bowel diseases (IBD). Novel risk variants in the Toll-like receptor 2 (TLR2) gene have previously been associated with a more severe disease phenotype in a subgroup of IBD patients. Recent studies have provided important insights of the commensal and host defense mechanisms to maintain functional barrier integrity of the intestinal epithelium through TLR2. Deficient TLR2 signaling may imbalance commensal-dependent intestinal epithelial barrier defense, facilitating mucosal injury and leading to increased susceptibility of colitis. Treatment with a synthetic TLR2 ligand significantly suppresses mucosal inflammation by efficiently protecting tight junction-associated integrity of the intestinal epithelium in vivo. These beneficial effects may be supplemented by TLR2-induced anti-inflammatory immune responses (such as interleukin-10 production) in lamina propria mononuclear cells. Thus, cell-specific TLR2 targeting may offer a novel therapeutic approach to human IBD therapy by protecting IEC barrier function.

The Chromatin Assembly Factor Complex 1 (CAF1) and 5-Azacytidine (5-AzaC) Affect Cell Motility in Src-transformed Human Epithelial Cells.

PubMed

Endo, Akinori; Ly, Tony; Pippa, Raffaella; Bensaddek, Dalila; Nicolas, Armel; Lamond, Angus I

2017-01-06

Tumor invasion into surrounding stromal tissue is a hallmark of high grade, metastatic cancers. Oncogenic transformation of human epithelial cells in culture can be triggered by activation of v-Src kinase, resulting in increased cell motility, invasiveness, and tumorigenicity and provides a valuable model for studying how changes in gene expression cause cancer phenotypes. Here, we show that epithelial cells transformed by activated Src show increased levels of DNA methylation and that the methylation inhibitor 5-azacytidine (5-AzaC) potently blocks the increased cell motility and invasiveness induced by Src activation. A proteomic screen for chromatin regulators acting downstream of activated Src identified the replication-dependent histone chaperone CAF1 as an important factor for Src-mediated increased cell motility and invasion. We show that Src causes a 5-AzaC-sensitive decrease in both mRNA and protein levels of the p150 (CHAF1A) and p60 (CHAF1B), subunits of CAF1. Depletion of CAF1 in untransformed epithelial cells using siRNA was sufficient to recapitulate the increased motility and invasive phenotypes characteristic of transformed cells without activation of Src. Maintaining high levels of CAF1 by exogenous expression suppressed the increased cell motility and invasiveness phenotypes when Src was activated. These data identify a critical role of CAF1 in the dysregulation of cell invasion and motility phenotypes seen in transformed cells and also highlight an important role for epigenetic remodeling through DNA methylation for Src-mediated induction of cancer phenotypes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Crystalline structure of pulverized dental calculus induces cell death in oral epithelial cells.

PubMed

Ziauddin, S M; Yoshimura, A; Montenegro Raudales, J L; Ozaki, Y; Higuchi, K; Ukai, T; Kaneko, T; Miyazaki, T; Latz, E; Hara, Y

2018-06-01

Dental calculus is a mineralized deposit attached to the tooth surface. We have shown that cellular uptake of dental calculus triggers nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation, leading to the processing of the interleukin-1β precursor into its mature form in mouse and human phagocytes. The activation of the NLRP3 inflammasome also induced a lytic form of programmed cell death, pyroptosis, in these cells. However, the effects of dental calculus on other cell types in periodontal tissue have not been investigated. The aim of this study was to determine whether dental calculus can induce cell death in oral epithelial cells. HSC-2 human oral squamous carcinoma cells, HOMK107 human primary oral epithelial cells and immortalized mouse macrophages were exposed to dental calculus or 1 of its components, hydroxyapatite crystals. For inhibition assays, the cells were exposed to dental calculus in the presence or absence of cytochalasin D (endocytosis inhibitor), z-YVAD-fmk (caspase-1 inhibitor) or glyburide (NLRP3 inflammasome inhibitor). Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release and staining with propidium iodide. Tumor necrosis factor-α production was quantified by enzyme-linked immunosorbent assay. Oral epithelial barrier function was examined by permeability assay. Dental calculus induced cell death in HSC-2 cells, as judged by LDH release and propidium iodide staining. Dental calculus also induced LDH release from HOMK107 cells. Following heat treatment, dental calculus lost its capacity to induce tumor necrosis factor-α in mouse macrophages, but could induce LDH release in HSC-2 cells, indicating a major role of inorganic components in cell death. Hydroxyapatite crystals also induced cell death in both HSC-2 and HOMK107 cells, as judged by LDH release, indicating the capacity of crystal particles to induce cell death. Cell death induced by dental calculus was significantly inhibited by cytochalasin D, z-YVAD-fmk and glyburide, indicating NLRP3 inflammasome involvement. In permeability assays, dental calculus attenuated the barrier function of HSC-2 cell monolayers. Dental calculus induces pyroptotic cell death in human oral epithelial cells and the crystalline structure plays a major role in this process. Oral epithelial cell death induced by dental calculus might be important for the etiology of periodontitis. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Barrier Epithelial Cells and the Control of Type 2 Immunity.

PubMed

Hammad, Hamida; Lambrecht, Bart N

2015-07-21

Type-2-cell-mediated immunity, rich in eosinophils, basophils, mast cells, CD4(+) T helper 2 (Th2) cells, and type 2 innate lymphoid cells (ILC2s), protects the host from helminth infection but also drives chronic allergic diseases like asthma and atopic dermatitis. Barrier epithelial cells (ECs) represent the very first line of defense and express pattern recognition receptors to recognize type-2-cell-mediated immune insults like proteolytic allergens or helminths. These ECs mount a prototypical response made up of chemokines, innate cytokines such as interleukin-1 (IL-1), IL-25, IL-33, and thymic stromal lymphopoietin (TSLP), as well as the alarmins uric acid, ATP, HMGB1, and S100 proteins. These signals program dendritic cells (DCs) to mount Th2-cell-mediated immunity and in so doing boost ILC2, basophil, and mast cell function. Here we review the general mechanisms of how different stimuli trigger type-2-cell-mediated immunity at mucosal barriers and how this leads to protection or disease. Copyright © 2015 Elsevier Inc. All rights reserved.

Lung stem cell differentiation in mice directed by endothelial cells via a BMP4-NFATc1-Thrombospondin-1 axis

PubMed Central

Lee, Joo-Hyeon; Bhang, Dong Ha; Beede, Alexander; Huang, Tian Lian; Stripp, Barry R.; Bloch, Kenneth D.; Wagers, Amy J.; Tseng, Yu-Hua; Ryeom, Sandra; Kim, Carla F.

2014-01-01

SUMMARY Lung stem cells are instructed to produce lineage-specific progeny through unknown factors in their microenvironment. We used clonal three-dimensional (3D) co-cultures of endothelial cells and distal lung stem cells, bronchioalveolar stem cells (BASCs), to probe the instructive mechanisms. Single BASCs had bronchiolar and alveolar differentiation potential in lung endothelial cell co-cultures. Gain and loss of function experiments showed BMP4-Bmpr1a signaling triggers calcineurin/NFATc1-dependent expression of Thrombospondin-1 (Tsp1) in lung endothelial cells to drive alveolar lineage-specific BASC differentiation. Tsp1-null mice exhibited defective alveolar injury repair, confirming a crucial role for the BMP4-NFATc1-TSP1 axis in lung epithelial differentiation and regeneration in vivo. Discovery of this pathway points to methods to direct the derivation of specific lung epithelial lineages from multipotent cells. These findings elucidate a pathway that may be a critical target in lung diseases and provide new tools to understand the mechanisms of respiratory diseases at the single cell level. PMID:24485453

Trichomonas vaginalis α-Actinin 2 Modulates Host Immune Responses by Inducing Tolerogenic Dendritic Cells via IL-10 Production from Regulatory T Cells.

PubMed

Lee, Hye-Yeon; Kim, Juri; Ryu, Jae-Sook; Park, Soon-Jung

2017-08-01

Trichomonas vaginalis is a pathogen that triggers severe immune responses in hosts. T. vaginalis α-actinin 2, Tvα-actinin 2, has been used to diagnose trichomoniasis. This study was undertaken to examine the role of Tvα-actinin 2 as an antigenic molecule to induce immune responses from humans. Western blot analysis using anti-Tvα-actinin 2 antibodies indicated its presence in the secreted proteins of T. vaginalis. ELISA was employed to measure cytokine production by vaginal epithelial cells, prostate cells, mouse dendritic cells (DCs), or T cells stimulated with T. vaginalis or Tvα-actinin 2 protein. Both T. vaginalis and rTvα-actinin 2 induced cytokine production from epithelial cell lines, including IL-10. Moreover, CD4+CD25- regulatory T cells (Treg cells) incubated with rTvα-actinin 2-treated DCs produced high levels of IL-10. These data indicate that Tvα-actinin 2 modulates immune responses via IL-10 production by Treg cells.

Sterigmatocystin induces G1 arrest in primary human esophageal epithelial cells but induces G2 arrest in immortalized cells: key mechanistic differences in these two models.

PubMed

Wang, Juan; Huang, Shujuan; Xing, Lingxiao; Cui, Jinfeng; Tian, Ziqiang; Shen, Haitao; Jiang, Xiujuan; Yan, Xia; Wang, Junling; Zhang, Xianghong

2015-11-01

Sterigmatocystin (ST), a mycotoxin commonly found in food and feed commodities, has been classified as a "possible human carcinogen." Our previous studies suggested that ST exposure might be a risk factor for esophageal cancer and that ST may induce DNA damage and G2 phase arrest in immortalized human esophageal epithelial cells (Het-1A). To further confirm and explore the cellular responses of ST in human esophageal epithelia, we comparatively evaluated DNA damage, cell cycle distribution and the relative mechanisms in primary cultured human esophageal epithelial cells (EPC), which represent a more representative model of the in vivo state, and Het-1A cells. In this study, we found that ST could induce DNA damage in both EPC and Het-1A cells but led to G1 phase arrest in EPC cells and G2 phase arrest in Het-1A cells. Furthermore, our results indicated that the activation of the ATM-Chk2 pathway was involved in ST-induced G1 phase arrest in EPC cells, whereas the p53-p21 pathway activation in ST-induced G2 phase arrest in Het-1A cells. Studies have demonstrated that SV40 large T-antigen (SV40LT) may disturb cell cycle progression by inactivating some of the proteins involved in the G1/S checkpoint. Het-1A is a non-cancerous epithelial cell line immortalized by SV40LT. To evaluate the possible perturbation effect of SV40LT on ST-induced cell cycle disturbance in Het-1A cells, we knocked down SV40LT of Het-1A cells with siRNA and found that under this condition, ST-induced G2 arrest was significantly attenuated, whereas the proportion of cells in the G1 phase was significantly increased. Furthermore, SV40LT-siRNA also inhibited the activation of the p53-p21 signaling pathway induced by ST. In conclusion, our data indicated that ST could induce DNA damage in both primary cultured and immortalized esophageal epithelial cells. In primary human esophageal epithelial cells, ST induced DNA damage and then triggered the ATM-Chk2 pathway, resulting in G1 phase arrest, whereas in SV40LT-immortalized human esophageal epithelial cells, SV40LT-mediated G1 checkpoint inactivation occurred, and ST-DNA damage activated p53-p21 signaling pathway, up-regulating G2/M phase regulatory proteins and finally leading to a G2 phase arrest. Thus, the SV40LT-mediated G1 checkpoint inactivation is responsible for the difference in the cell cycle arrest by ST between immortalized and primary cultured human esophageal epithelial cells.

Pseudomonas aeruginosa biofilm-associated homoserine lactone C12 rapidly activates apoptosis in airway epithelia

PubMed Central

Schwarzer, Christian; Fu, Zhu; Patanwala, Maria; Hum, Lauren; Lopez-Guzman, Mirielle; Illek, Beate; Kong, Weidong; Lynch, Susan V.; Machen, Terry E.

2014-01-01

Pseudomonas aeruginosa (PA) forms biofilms in lungs of cystic fibrosis CF) patients, a process regulated by quorum sensing molecules including N-(3-oxododecanoyl)-L-homoserine lactone, C12. C12 (10–100 μM) rapidly triggered events commonly associated with the intrinsic apoptotic pathway in JME (CFΔF508CFTR, nasal surface) epithelial cells: depolarization of mitochondrial (mito) membrane potential (Δψmito) and release of cytochrome C (cytoC) from mitos into cytosol and activation of caspases 3/7, 8 and 9. C12 also had novel effects on the endoplasmic reticulum (release of both Ca2+ and ER-targeted GFP and oxidized contents into the cytosol). Effects began within 5 minutes and were complete in 1–2 hrs. C12 caused similar activation of caspases and release of cytoC from mitos in Calu-3 (wtCFTR, bronchial gland) cells, showing that C12-triggered responses occurred similarly in different airway epithelial types. C12 had nearly identical effects on three key aspects of the apoptosis response (caspase 3/7, depolarization of Δψmito and reduction of redox potential in the ER) in JME and CFTR-corrected JME cells (adenoviral expression), showing that CFTR was likely not an important regulator of C12-triggered apoptosis in airway epithelia. Exposure of airway cultures to biofilms from PAO1wt caused depolarization of Δψmito and increases in Cacyto like 10–50 μM C12. In contrast, biofilms from PAO1ΔlasI (C12 deficient) had no effect, suggesting that C12 from P. aeruginosa biofilms may contribute to accumulation of apoptotic cells that cannot be cleared from CF lungs. A model to explain the effects of C12 is proposed. PMID:22233488

BRCA1/p220 loss triggers BRCA1-IRIS overexpression via mRNA stabilization in breast cancer cells

PubMed Central

Shimizu, Yoshiko; Mullins, Nicole; Blanchard, Zannel; ElShamy, Wael M.

2012-01-01

BRCA1/p220-assocaited and triple negative/basal-like (TN/BL) tumors are aggressive and incurable breast cancer diseases that share among other features the no/low BRCA1/p220 expression. Here we show that BRCA1/p220 silencing in normal human mammary epithelial (HME) cells reduces expression of two RNA-destabilizing proteins, namely AUF1 and pCBP2, both proteins bind and destabilize BRCA1-IRIS mRNA. BRCA1-IRIS overexpression in HME cells triggers expression of several TN/BL markers, e.g., cytokeratins 5 and 17, p-cadherin, EGFR and cyclin E as well as expression and activation of the pro-survival proteins; AKT and survivin. BRCA1-IRIS silencing in the TN/BL cell line, SUM149 or restoration of BRCA1/p220 expression in the mutant cell line, HCC1937 reduced expression of TN/BL markers, AKT, survivin, and induced cell death. Collectively, we propose that BRCA1/p220 loss of expression or function triggers BRCA1-IRIS overexpression through a post-transcriptional mechanism, which in turn promotes formation of aggressive and invasive breast tumors by inducing expression of TN/BL and survival proteins. PMID:22431556

Matriptase activation connects tissue factor-dependent coagulation initiation to epithelial proteolysis and signaling.

PubMed

Le Gall, Sylvain M; Szabo, Roman; Lee, Melody; Kirchhofer, Daniel; Craik, Charles S; Bugge, Thomas H; Camerer, Eric

2016-06-23

The coagulation cascade is designed to sense tissue injury by physical separation of the membrane-anchored cofactor tissue factor (TF) from inactive precursors of coagulation proteases circulating in plasma. Once TF on epithelial and other extravascular cells is exposed to plasma, sequential activation of coagulation proteases coordinates hemostasis and contributes to host defense and tissue repair. Membrane-anchored serine proteases (MASPs) play critical roles in the development and homeostasis of epithelial barrier tissues; how MASPs are activated in mature epithelia is unknown. We here report that proteases of the extrinsic pathway of blood coagulation transactivate the MASP matriptase, thus connecting coagulation initiation to epithelial proteolysis and signaling. Exposure of TF-expressing cells to factors (F) VIIa and Xa triggered the conversion of latent pro-matriptase to an active protease, which in turn cleaved the pericellular substrates protease-activated receptor-2 (PAR2) and pro-urokinase. An activation pathway-selective PAR2 mutant resistant to direct cleavage by TF:FVIIa and FXa was activated by these proteases when cells co-expressed pro-matriptase, and matriptase transactivation was necessary for efficient cleavage and activation of wild-type PAR2 by physiological concentrations of TF:FVIIa and FXa. The coagulation initiation complex induced rapid and prolonged enhancement of the barrier function of epithelial monolayers that was dependent on matriptase transactivation and PAR2 signaling. These observations suggest that the coagulation cascade engages matriptase to help coordinate epithelial defense and repair programs after injury or infection, and that matriptase may contribute to TF-driven pathogenesis in cancer and inflammation.

Berberine activates Nrf2 nuclear translocation and inhibits apoptosis induced by high glucose in renal tubular epithelial cells through a phosphatidylinositol 3-kinase/Akt-dependent mechanism.

PubMed

Zhang, Xiuli; Liang, Dan; Lian, Xu; Jiang, Yan; He, Hui; Liang, Wei; Zhao, Yue; Chi, Zhi-Hong

2016-06-01

Apoptosis of tubular epithelial cells is a major feature of diabetic kidney disease, and hyperglycemia triggers the generation of free radicals and oxidant stress in tubular cells. Berberine (BBR) is identified as a potential anti-diabetic herbal medicine due to its beneficial effects on insulin sensitivity, glucose metabolism and glycolysis. In this study, the underlying mechanisms involved in the protective effects of BBR on high glucose-induced apoptosis were explored using cultured renal tubular epithelial cells (NRK-52E cells) and human kidney proximal tubular cell line (HK-2 cells). We identified the pivotal role of phosphatidylinositol 3-kinase (PI3K)/Akt in BBR cellular defense mechanisms and revealed the novel effect of BBR on nuclear factor (erythroid-derived 2)-related factor-2 (Nrf2) and heme oxygenase (HO)-1 in NRK-52E and HK-2 cells. BBR attenuated reactive oxygen species production, antioxidant defense (GSH and SOD) and oxidant-sensitive proteins (Nrf2 and HO-1), which also were blocked by LY294002 (an inhibitor of PI3K) in HG-treated NRK-52E and HK-2 cells. Furthermore, BBR improved mitochondrial function by increasing mitochondrial membrane potential. BBR-induced anti-apoptotic function was demonstrated by decreasing apoptotic proteins (cytochrome c, Bax, caspase3 and caspase9). All these findings suggest that BBR exerts the anti-apoptosis effects through activation of PI3K/Akt signal pathways and leads to activation of Nrf2 and induction of Nrf2 target genes, and consequently protecting the renal tubular epithelial cells from HG-induced apoptosis.

Novel triazolothiadiazines act as potent anticancer agents in liver cancer cells through Akt and ASK-1 proteins.

PubMed

Aytaç, Peri S; Durmaz, Irem; Houston, Douglas R; Çetin-Atalay, Rengül; Tozkoparan, Birsen

2016-02-15

Newly designed triazolothiadiazines incorporating with structural motifs of nonsteroidal analgesic anti-inflammatory drugs were synthesized and screened for their bioactivity against epithelial cancer cells. Compounds with bioactivities less then ∼5μM (IC50) were further analyzed and showed to induce apoptotic cell death and SubG1 cell cycle arrest in liver cancer cells. Among this group, two compounds (1g and 1h) were then studied to identify the mechanism of action. These molecules triggered oxidative stress induced apoptosis through ASK-1 protein activation and Akt protein inhibition as demonstrated by downstream targets such as GSK3β, β-catenin and cyclin D1. QSAR and molecular docking models provide insight into the mechanism of inhibition and indicate the optimal direction of future synthetic efforts. Furthermore, molecular docking results were confirmed with in vitro COX bioactivity studies. This study demonstrates that the novel triazolothiadiazine derivatives are promising drug candidates for epithelial cancers, especially liver cancer. Copyright © 2016. Published by Elsevier Ltd.

Estrogen-related receptor α participates transforming growth factor-β (TGF-β) induced epithelial-mesenchymal transition of osteosarcoma cells

PubMed Central

Chen, Yantao; Zhang, Kunshui; Li, Yang; He, Qing

2017-01-01

ABSTRACT Osteosarcoma patients often exhibit pulmonary metastasis, which results in high patient mortality. Understanding the mechanisms of advanced metastasis in osteosarcoma cell is important for the targeted treatment and drug development. Our present study revealed that transforming growth factor-β (TGF-β) treatment can significantly promote the in vitro migration and invasion of human osteosarcoma MG-63 and HOS cells. The loss of epithelial characteristics E-cadherin (E-Cad) and up regulation of mesenchymal markers Vimentin (Vim) suggested TGF-β induced epithelial-mesenchymal transition (EMT) of osteosarcoma cells. TGF-β treatment obviously increased the expression of Snail, a key EMT-related transcription factor, in both MG-63 and HOS cells. Silencing of Snail markedly attenuated TGF-β induced down regulation of E-cad and up regulation of Vim. TGF-β treatment also significantly increased the expression and nuclear translocation of estrogen-related receptors α (ERRα), while had no obvious effect on the expression of ERα, ERβ, or ERRγ. Knock down of ERRα or its inhibitor XCT-790 significantly attenuated TFG-β induced EMT and transcription of Snail in osteosarcoma cells. Collectively, our present study revealed that TGF-β treatment can trigger the EMT of osteosarcoma cells via ERRα/Snail pathways. Our data suggested that ERRα/Snail pathways might be potential therapeutic targets of metastasis of osteosarcoma cells. PMID:27532429

Perfluorocarbon induces alveolar epithelial cell response through structural and mechanical remodeling.

PubMed

André Dias, Sofia; Planus, Emmanuelle; Angely, Christelle; Lotteau, Luc; Tissier, Renaud; Filoche, Marcel; Louis, Bruno; Pelle, Gabriel; Isabey, Daniel

2018-02-15

During total liquid ventilation, lung cells are exposed to perfluorocarbon (PFC) whose chemophysical properties highly differ from standard aqueous cell feeding medium (DMEM). We herein perform a systematic study of structural and mechanical properties of A549 alveolar epithelial cells in order to characterize their response to PFC exposure, using DMEM as control condition. Changes in F-actin structure, focal adhesion density and glycocalyx distribution are evaluated by confocal fluorescent microscopy. Changes in cell mechanics and adhesion are measured by multiscale magnetic twisting cytometry (MTC). Two different microrheological models (single Voigt and power law) are used to analyze the cell mechanics characterized by cytoskeleton (CSK) stiffness and characteristic relaxation times. Cell-matrix adhesion is analyzed using a stochastic multibond deadhesion model taking into account the non-reversible character of the cell response, allowing us to quantify the adhesion weakness and the number of associated bonds. The roles of F-actin structure and glycocalyx layer are evaluated by depolymerizing F-actin and degrading glycocalyx, respectively. Results show that PFC exposure consistently induces F-actin remodeling, CSK softening and adhesion weakening. These results demonstrate that PFC triggers an alveolar epithelial cell response herein evidenced by a decay in intracellular CSK tension, an adhesion weakening and a glycocalyx layer redistribution. These PFC-induced cell adjustments are consistent with the hypothesis that cells respond to a decrease in adhesion energy at cell surface. This adhesion energy can be even further reduced in the presence of surfactant adsorbed at the cell surface.

Analysis of the Oxidative Stress Status in Nonspecific Vaginitis and Its Role in Vaginal Epithelial Cells Apoptosis

PubMed Central

Chen, Zhaojie; Zhang, Zhen; Zhang, Haiyan; Xie, Beibei

2015-01-01

Nonspecific vaginitis (NSV), also named bacterial vaginosis, is one of the most common genital system diseases in women during their reproductive years. The specific pathogenic mechanism of NSV is not clear yet. Upon the balance alteration, large amount of reactive oxidant species (ROS) is generated and accumulated in the genital tract, and thus resulting in oxidative stress, which has been reported to be an important trigger of mitochondrial pathway cell apoptosis. In this study, the antioxidant secretion level and antioxidant enzyme activity in the vaginal discharge were evaluated to analyze the oxidative status in the vaginal tract of NSV patients. The effect of oxidative stress on the vaginal mucosa epithelial cell apoptosis was then studied. The role of oxidative stress on NSV development was uncovered; thus open new direction for the prevention and treatment of NSV by providing antiradical agents was revealed. PMID:26558281

Cell adhesion heterogeneity reinforces tumour cell dissemination: novel insights from a mathematical model.

PubMed

Reher, David; Klink, Barbara; Deutsch, Andreas; Voss-Böhme, Anja

2017-08-11

Cancer cell invasion, dissemination, and metastasis have been linked to an epithelial-mesenchymal transition (EMT) of individual tumour cells. During EMT, adhesion molecules like E-cadherin are downregulated and the decrease of cell-cell adhesion allows tumour cells to dissociate from the primary tumour mass. This complex process depends on intracellular cues that are subject to genetic and epigenetic variability, as well as extrinsic cues from the local environment resulting in a spatial heterogeneity in the adhesive phenotype of individual tumour cells. Here, we use a novel mathematical model to study how adhesion heterogeneity, influenced by intrinsic and extrinsic factors, affects the dissemination of tumour cells from an epithelial cell population. The model is a multiscale cellular automaton that couples intracellular adhesion receptor regulation with cell-cell adhesion. Simulations of our mathematical model indicate profound effects of adhesion heterogeneity on tumour cell dissemination. In particular, we show that a large variation of intracellular adhesion receptor concentrations in a cell population reinforces cell dissemination, regardless of extrinsic cues mediated through the local cell density. However, additional control of adhesion receptor concentration through the local cell density, which can be assumed in healthy cells, weakens the effect. Furthermore, we provide evidence that adhesion heterogeneity can explain the remarkable differences in adhesion receptor concentrations of epithelial and mesenchymal phenotypes observed during EMT and might drive early dissemination of tumour cells. Our results suggest that adhesion heterogeneity may be a universal trigger to reinforce cell dissemination in epithelial cell populations. This effect can be at least partially compensated by a control of adhesion receptor regulation through neighbouring cells. Accordingly, our findings explain how both an increase in intra-tumour adhesion heterogeneity and the loss of control through the local environment can promote tumour cell dissemination. This article was reviewed by Hanspeter Herzel, Thomas Dandekar and Marek Kimmel.

Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish.

PubMed

Chenais, Nathalie; Lareyre, Jean-Jacques; Le Bail, Pierre-Yves; Labbe, Catherine

2015-07-01

The development of fin primary cell cultures for in vitro cellular and physiological studies is hampered by slow cell outgrowth, low proliferation rate, poor viability, and sparse cell characterization. Here, we investigated whether the recycling of fresh explants after a first conventional culture could improve physiological stability and sustainability of the culture. The recycled explants were able to give a supplementary cell culture showing faster outgrowth, cleaner cell layers and higher net cell production. The cells exhibited a highly stabilized profile for marker gene expression including a low cytokeratin 49 (epithelial marker) and a high collagen 1a1 (mesenchymal marker) expression. Added to the cell spindle-shaped morphology, motility behavior, and actin organization, this suggests that the cells bore stable mesenchymal characteristics. This contrast with the time-evolving expression pattern observed in the control fresh explants during the first 2 weeks of culture: a sharp decrease in cytokeratin 49 expression was concomitant with a gradual increase in col1a1. We surmise that such loss of epithelial features for the benefit of mesenchymal ones was triggered by an epithelial to mesenchymal transition (EMT) process or by way of a progressive population replacement process. Overall, our findings provide a comprehensive characterization of this new primary culture model bearing mesenchymal features and whose stability over culture time makes those cells good candidates for cell reprogramming prior to nuclear transfer, in a context of fish genome preservation. Copyright © 2015 Elsevier Inc. All rights reserved.

Synergic activation of toll-like receptor (TLR) 2/6 and 9 in response to Ureaplasma parvum & urealyticum in human amniotic epithelial cells.

PubMed

Triantafilou, Martha; De Glanville, Benjamin; Aboklaish, Ali F; Spiller, O Brad; Kotecha, Sailesh; Triantafilou, Kathy

2013-01-01

Ureaplasma species are the most frequently isolated microorganisms inside the amniotic cavity and have been associated with spontaneous abortion, chorioamnionitis, premature rupture of the membranes (PROM), preterm labour (PL) pneumonia in neonates and bronchopulmonary dysplasia in neonates. The mechanisms by which Ureaplasmas cause such diseases remain unclear, but it is believed that inappropriate induction of inflammatory responses is involved, triggered by the innate immune system. As part of its mechanism of activation, the innate immune system employs germ-lined encoded receptors, called pattern recognition receptors (PRRs) in order to "sense" pathogens. One such family of PRRs are the Toll like receptor family (TLR). In the current study we aimed to elucidate the role of TLRs in Ureaplasma-induced inflammation in human amniotic epithelial cells. Using silencing, as well as human embryonic kidney (HEK) transfected cell lines, we demonstrate that TLR2, TLR6 and TLR9 are involved in the inflammatory responses against Ureaplasma parvum and urealyticum serovars. Ureaplasma lipoproteins, such as Multiple Banded antigen (MBA), trigger responses via TLR2/TLR6, whereas the whole bacterium is required for TLR9 activation. No major differences were observed between the different serovars. Cell activation by Ureaplasma parvum and urealyticum seem to require lipid raft function and formation of heterotypic receptor complexes comprising of TLR2 and TLR6 on the cell surface and TLR9 intracellularly.

Synergic Activation of Toll-Like Receptor (TLR) 2/6 and 9 in Response to Ureaplasma parvum & urealyticum in Human Amniotic Epithelial Cells

PubMed Central

Triantafilou, Martha; De Glanville, Benjamin; Aboklaish, Ali F.; Spiller, O. Brad; Kotecha, Sailesh; Triantafilou, Kathy

2013-01-01

Ureaplasma species are the most frequently isolated microorganisms inside the amniotic cavity and have been associated with spontaneous abortion, chorioamnionitis, premature rupture of the membranes (PROM), preterm labour (PL) pneumonia in neonates and bronchopulmonary dysplasia in neonates. The mechanisms by which Ureaplasmas cause such diseases remain unclear, but it is believed that inappropriate induction of inflammatory responses is involved, triggered by the innate immune system. As part of its mechanism of activation, the innate immune system employs germ-lined encoded receptors, called pattern recognition receptors (PRRs) in order to “sense” pathogens. One such family of PRRs are the Toll like receptor family (TLR). In the current study we aimed to elucidate the role of TLRs in Ureaplasma-induced inflammation in human amniotic epithelial cells. Using silencing, as well as human embryonic kidney (HEK) transfected cell lines, we demonstrate that TLR2, TLR6 and TLR9 are involved in the inflammatory responses against Ureaplasma parvum and urealyticum serovars. Ureaplasma lipoproteins, such as Multiple Banded antigen (MBA), trigger responses via TLR2/TLR6, whereas the whole bacterium is required for TLR9 activation. No major differences were observed between the different serovars. Cell activation by Ureaplasma parvum and urealyticum seem to require lipid raft function and formation of heterotypic receptor complexes comprising of TLR2 and TLR6 on the cell surface and TLR9 intracellularly. PMID:23593431

Phenotypic chemical screening using a zebrafish neural crest EMT reporter identifies retinoic acid as an inhibitor of epithelial morphogenesis

PubMed Central

Jimenez, Laura; Wang, Jindong; Morrison, Monique A.; Whatcott, Clifford; Soh, Katherine K.; Warner, Steven; Bearss, David; Jette, Cicely A.; Stewart, Rodney A.

2016-01-01

ABSTRACT The epithelial-to-mesenchymal transition (EMT) is a highly conserved morphogenetic program essential for embryogenesis, regeneration and cancer metastasis. In cancer cells, EMT also triggers cellular reprogramming and chemoresistance, which underlie disease relapse and decreased survival. Hence, identifying compounds that block EMT is essential to prevent or eradicate disseminated tumor cells. Here, we establish a whole-animal-based EMT reporter in zebrafish for rapid drug screening, called Tg(snai1b:GFP), which labels epithelial cells undergoing EMT to produce sox10-positive neural crest (NC) cells. Time-lapse and lineage analysis of Tg(snai1b:GFP) embryos reveal that cranial NC cells delaminate from two regions: an early population delaminates adjacent to the neural plate, whereas a later population delaminates from within the dorsal neural tube. Treating Tg(snai1b:GFP) embryos with candidate small-molecule EMT-inhibiting compounds identified TP-0903, a multi-kinase inhibitor that blocked cranial NC cell delamination in both the lateral and medial populations. RNA sequencing (RNA-Seq) analysis and chemical rescue experiments show that TP-0903 acts through stimulating retinoic acid (RA) biosynthesis and RA-dependent transcription. These studies identify TP-0903 as a new therapeutic for activating RA in vivo and raise the possibility that RA-dependent inhibition of EMT contributes to its prior success in eliminating disseminated cancer cells. PMID:26794130

Recurrent exposure to nicotine differentiates human bronchial epithelial cells via epidermal growth factor receptor activation

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

Martinez-Garcia, Eva; Irigoyen, Marta; Anso, Elena

Cigarette smoking is the major preventable cause of lung cancer in developed countries. Nicotine (3-(1-methyl-2-pyrrolidinyl)-pyridine) is one of the major alkaloids present in tobacco. Besides its addictive properties, its effects have been described in panoply of cell types. In fact, recent studies have shown that nicotine behaves as a tumor promoter in transformed epithelial cells. This research focuses on the effects of acute repetitive nicotine exposure on normal human bronchial epithelial cells (NHBE cells). Here we show that treatment of NHBE cells with recurrent doses of nicotine up to 500 {mu}M triggered cell differentiation towards a neuronal-like phenotype: cells emittedmore » filopodia and expressed neuronal markers such as neuronal cell adhesion molecule, neurofilament-M and the transcription factors neuronal N and Pax-3. We also demonstrate that nicotine treatment induced NF-kB translocation to the nucleus, phosphorylation of the epidermal growth factor receptor (EGFR), and accumulation of heparin binding-EGF in the extracellular medium. Moreover, addition of AG1478, an inhibitor of EGFR tyrosine phosphorylation, or cetuximab, a monoclonal antibody that precludes ligand binding to the same receptor, prevented cell differentiation by nicotine. Lastly, we show that differentiated cells increased their adhesion to the extracellular matrix and their protease activity. Given that several lung pathologies are strongly related to tobacco consumption, these results may help to better understand the damaging consequences of nicotine exposure.« less

High LET Radiation Can Enhance TGF(Beta) Induced EMT and Cross-Talk with ATM Pathways

NASA Technical Reports Server (NTRS)

Wang, Minli; Hada, Megumi; Huff, Janice; Pluth, Janice M.; Anderson, Janniffer; ONeill, Peter; Cucinotta, Francis A.

2010-01-01

The TGF(Beta) pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation in mammary epithelial cells. We investigated possible interactions between the TGF(Beta) and ATM pathways following simulated space radiation using hTERT immortalized human esophageal epithelial cells (EPC-hTERT), mink lung epithelial cells (Mv1lu), and several human fibroblast cell lines. TGF(Beta) is a key modulator of the Epithelial-Mesenchymal Transition (EMT), important in cancer progression and metastasis. The implication of EMT by radiation also has several lines of developing evidence, however is poorly understood. The identification of TGF(Beta) induced EMT can be shown in changes to morphology, related gene over expression or down regulation, which can be detected by RT-PCR, and immunostaining and western blotting. In this study, we have observed morphologic and molecular alternations consistent with EMT after Mv1lu cells were treated with TGF(Beta) High LET radiation enhanced TGF(Beta) mediated EMT with a dose as low as 0.1Gy. In order to consider the TGF(Beta) interaction with ATM we used a potent ATM inhibitor Ku55933 and investigated gene expression changes and Smad signaling kinetics. Ku559933 was observed to reverse TGF(Beta) induced EMT, while this was not observed in dual treated cells (radiation+TGF(Beta)). In EPC-hTERT cells, TGF(Beta) alone was not able to induce EMT after 3 days of application. A combined treatment with high LET, however, significantly caused the alteration of EMT markers. To study the function of p53 in the process of EMT, we knocked down P53 through RNA interference. Morphology changes associated with EMT were observed in epithelial cells with silenced p53. Our study indicates: high LET radiation can enhance TGF(Beta) induced EMT; while ATM is triggering the process of TGF(Beta)-induced EMT, p53 might be an essential repressor for EMT phenotypes.

Colon cancer-associated B2 Escherichia coli colonize gut mucosa and promote cell proliferation

PubMed Central

Raisch, Jennifer; Buc, Emmanuel; Bonnet, Mathilde; Sauvanet, Pierre; Vazeille, Emilie; de Vallée, Amélie; Déchelotte, Pierre; Darcha, Claude; Pezet, Denis; Bonnet, Richard; Bringer, Marie-Agnès; Darfeuille-Michaud, Arlette

2014-01-01

AIM: To provide further insight into the characterization of mucosa-associated Escherichia coli (E. coli) isolated from the colonic mucosa of cancer patients. METHODS: Phylogroups and the presence of cyclomodulin-encoding genes of mucosa-associated E. coli from colon cancer and diverticulosis specimens were determined by PCR. Adhesion and invasion experiments were performed with I-407 intestinal epithelial cells using gentamicin protection assay. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) expression in T84 intestinal epithelial cells was measured by enzyme-linked immunosorbent assay and by Western Blot. Gut colonization, inflammation and pro-carcinogenic potential were assessed in a chronic infection model using CEABAC10 transgenic mice. Cell proliferation was analyzed by real-time mRNA quantification of PCNA and immunohistochemistry staining of Ki67. RESULTS: Analysis of mucosa-associated E. coli from colon cancer and diverticulosis specimens showed that whatever the origin of the E. coli strains, 86% of cyclomodulin-positive E. coli belonged to B2 phylogroup and most harbored polyketide synthase (pks) island, which encodes colibactin, and/or cytotoxic necrotizing factor (cnf) genes. In vitro assays using I-407 intestinal epithelial cells revealed that mucosa-associated B2 E. coli strains were poorly adherent and invasive. However, mucosa-associated B2 E. coli similarly to Crohn’s disease-associated E. coli are able to induce CEACAM6 expression in T84 intestinal epithelial cells. In addition, in vivo experiments using a chronic infection model of CEACAM6 expressing mice showed that B2 E. coli strain 11G5 isolated from colon cancer is able to highly persist in the gut, and to induce colon inflammation, epithelial damages and cell proliferation. CONCLUSION: In conclusion, these data bring new insights into the ability of E. coli isolated from patients with colon cancer to establish persistent colonization, exacerbate inflammation and trigger carcinogenesis. PMID:24914378

Osthole inhibits the tumorigenesis of hepatocellular carcinoma cells.

PubMed

Lin, Zhi-Kun; Liu, Jia; Jiang, Guo-Qiang; Tan, Guang; Gong, Peng; Luo, Hai-Feng; Li, Hui-Min; Du, Jian; Ning, Zhen; Xin, Yi; Wang, Zhong-Yu

2017-03-01

Hepatocellular carcinoma (HCC) accounts for approximately 90% of all cases of primary liver cancer, and the majority of patients with HCC are deprived of effective curative methods. Osthole is a Chinese herbal medicine which has been reported to possess various pharmacological functions, including hepatocellular protection. In the present study, we investigated the anticancer activity of osthole using HCC cell lines. We found that osthole inhibited HCC cell proliferation, induced cell cycle arrest, triggered DNA damage and suppressed migration in HCC cell lines. Furthermore, we demonstrated that osthole not only contributed to cell cycle G2/M phase arrest via downregulation of Cdc2 and cyclin B1 levels, but also induced DNA damage via an increase in ERCC1 expression. In addition, osthole inhibited the migration of HCC cell lines by significantly downregulating MMP-2 and MMP-9 levels. Finally, we demonstrated that osthole inhibited epithelial-mesenchymal transition (EMT) via increasing the expression of epithelial biomarkers E-cadherin and β-catenin, and significantly decreasing mesenchymal N-cadherin and vimentin protein expression. These results suggest that osthole may have potential chemotherapeutic activity against HCC.

Adenovirus E4ORF1-induced MYC activation promotes host cell anabolic glucose metabolism and virus replication.

PubMed

Thai, Minh; Graham, Nicholas A; Braas, Daniel; Nehil, Michael; Komisopoulou, Evangelia; Kurdistani, Siavash K; McCormick, Frank; Graeber, Thomas G; Christofk, Heather R

2014-04-01

Virus infections trigger metabolic changes in host cells that support the bioenergetic and biosynthetic demands of viral replication. Although recent studies have characterized virus-induced changes in host cell metabolism (Munger et al., 2008; Terry et al., 2012), the molecular mechanisms by which viruses reprogram cellular metabolism have remained elusive. Here, we show that the gene product of adenovirus E4ORF1 is necessary for adenovirus-induced upregulation of host cell glucose metabolism and sufficient to promote enhanced glycolysis in cultured epithelial cells by activation of MYC. E4ORF1 localizes to the nucleus, binds to MYC, and enhances MYC binding to glycolytic target genes, resulting in elevated expression of specific glycolytic enzymes. E4ORF1 activation of MYC promotes increased nucleotide biosynthesis from glucose intermediates and enables optimal adenovirus replication in primary lung epithelial cells. Our findings show how a viral protein exploits host cell machinery to reprogram cellular metabolism and promote optimal progeny virion generation. Copyright © 2014 Elsevier Inc. All rights reserved.

Lycium barbarum Polysaccharides Protect Rat Corneal Epithelial Cells against Ultraviolet B-Induced Apoptosis by Attenuating the Mitochondrial Pathway and Inhibiting JNK Phosphorylation.

PubMed

Du, Shaobo; Han, Biao; Li, Kang; Zhang, Xuan; Sha, Xueli; Gao, Lan

2017-01-01

Lycium barbarum polysaccharides (LBPs) have been shown to play a key role in protecting the eyes by reducing the apoptosis induced by certain types of damage. However, it is not known whether LBPs can protect damaged corneal cells from apoptosis. Moreover, no reports have focused on the role of LBPs in guarding against ultraviolet B- (UVB-) induced apoptosis. The present study aimed to investigate the protective effect and underlying mechanism of LBPs against UVB-induced apoptosis in rat corneal epithelial (RCE) cells. The results showed that LBPs significantly prevented the loss of cell viability and inhibited cell apoptosis induced by UVB in RCE cells. LBPs also inhibited UVB-induced loss of mitochondrial membrane potential, downregulation of Bcl-2 , and upregulation of Bax and caspase-3. Finally, LBPs attenuated the phosphorylation of c-Jun NH 2 -terminal kinase (JNK) triggered by UVB. In summary, LBPs protect RCE cells against UVB-induced damage and apoptosis, and the underlying mechanism involves the attenuation of the mitochondrial apoptosis pathway and the inhibition of JNK phosphorylation.

S1P1 receptor inhibits kidney epithelial mesenchymal transition triggered by ischemia/reperfusion injury via the PI3K/Akt pathway.

PubMed

Wang, Weina; Wang, Aimei; Luo, Guochang; Ma, Fengqiao; Wei, Xiaoming; Bi, Yongyi

2018-06-13

Ischemia/reperfusion (I/R) is a major cause of acute kidney injury (AKI), along with delayed graft function, which can trigger chronic kidney injury by stimulating epithelial to mesenchymal transition (EMT) in the kidney canaliculus. Sphingosine 1-phosphate receptor 1 (S1P1) is a G protein-coupled receptor that is indispensable for vessel homeostasis. This study aimed to investigate the influence of S1P1 on the mechanisms underlying I/R-induced EMT in the kidney using in vivo and in vitro models. Wild-type (WT) and S1P1-overexpressing kidney canaliculus cells were subject to hypoxic conditions followed by reoxygenation in the presence or absence of FTY720-P, a potent S1P1 agonist. In vivo, bilateral arteria renalis in wild-type mice and mice with silenced S1P1 were clamped for 30 min to obtain I/R models. We found that hypoxia/reoxygenation (H/R) significantly enhanced the expressions of EMT biomarkers and down-regulated S1P1 expression in wild-type canaliculus cells. In contrast, FTY720-P treatment or overexpression of S1P1 significantly suppressed EMT in wild-type canaliculus cells. Furthermore, after 48-72 h, a significant upregulation of EMT biomarker expression was triggered by I/R in mice with silenced S1P1, while the expressions of these markers did not change in wild-type mice. A kt activity was increased with H/R-induced EMT, suggesting that the protective influence of FTY720-P was due to its inhibition of PI3K/Akt. Therefore, the results of this study provide evidence that down-regulation of S1P1 expression is essential for the generation and progression of EMT triggered by I/R. S1P1 exhibits a prominent inhibitory effect on kidney I/R-induced EMT in the kidney by affecting the PI3K/Akt pathway.

Heparanase regulates the M1 polarization of renal macrophages and their crosstalk with renal epithelial tubular cells after ischemia/reperfusion injury.

PubMed

Masola, Valentina; Zaza, Gianluigi; Bellin, Gloria; Dall'Olmo, Luigi; Granata, Simona; Vischini, Gisella; Secchi, Maria Francesca; Lupo, Antonio; Gambaro, Giovanni; Onisto, Maurizio

2018-02-01

Heparanase (HPSE) is part of the biologic network triggered by ischemia/reperfusion (I/R) injury, a complication of renal transplantation and acute kidney injury. During this period, the kidney or graft undergoes a process of macrophages recruitment and activation. HPSE may therefore control these biologic effects. We measured the ability of HPSE and its inhibitor, SST0001, to regulate macrophage polarization and the crosstalk between macrophages and HK-2 renal tubular cells during in vitro hypoxia/reoxygenation (H/R). Furthermore, we evaluated in vivo renal inflammation, macrophage polarization, and histologic changes in mice subjected to monolateral I/R and treated with SST0001 for 2 or 7 d. The in vitro experiments showed that HPSE sustained M1 macrophage polarization and modulated apoptosis, the release of damage associated molecular patterns in post-H/R tubular cells, the synthesis of proinflammatory cytokines, and the up-regulation of TLRs on both epithelial cells and macrophages. HPSE also regulated M1 polarization induced by H/R-injured tubular cells and the partial epithelial-mesenchymal transition of these epithelial cells by M1 macrophages. All these effects were prevented by inhibiting HPSE. Furthermore, the inhibition of HPSE in vivo reduced inflammation and M1 polarization in mice undergoing I/R injury, partially restored renal function and normal histology, and reduced apoptosis. These results show for the first time that HPSE regulates macrophage polarization as well as renal damage and repair after I/R. HPSE inhibitors could therefore provide a new pharmacologic approach to minimize acute kidney injury and to prevent the chronic profibrotic damages induced by I/R.-Masola, V., Zaza, G., Bellin, G., Dall'Olmo, L., Granata, S., Vischini, G., Secchi, M. F., Lupo, A., Gambaro, G., Onisto, M. Heparanase regulates the M1 polarization of renal macrophages and their crosstalk with renal epithelial tubular cells after ischemia/reperfusion injury.

Border Forces and Friction Control Epithelial Closure Dynamics

PubMed Central

Cochet-Escartin, Olivier; Ranft, Jonas; Silberzan, Pascal; Marcq, Philippe

2014-01-01

We study the closure dynamics of a large number of well-controlled circular apertures within an epithelial monolayer, where the collective cell migration responsible for epithelization is triggered by the removal of a spatial constraint rather than by scratching. Based on experimental observations, we propose a physical model that takes into account border forces, friction with the substrate, and tissue rheology. Border protrusive activity drives epithelization despite the presence of a contractile actomyosin cable at the periphery of the wound. The closure dynamics is quantified by an epithelization coefficient, defined as the ratio of protrusive stress to tissue-substrate friction, that allows classification of different phenotypes. The same analysis demonstrates a distinct signature for human cells bearing the oncogenic RasV12 mutation, demonstrating the potential of the approach to quantitatively characterize metastatic transformations. PMID:24411238

Mitochondrial ATAD3A regulates milk biosynthesis and proliferation of mammary epithelial cells from dairy cow via the mTOR pathway.

PubMed

Chen, Dongying; Yuan, Xiaohan; Liu, Lijie; Zhang, Minghui; Qu, Bo; Zhen, Zhen; Gao, Xuejun

2018-05-01

ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane protein, which is essential for cell growth and metabolism. The mechanism by which ATAD3A acts is still not fully understood. In this study, we explored the regulatory role of ATAD3A on milk biosynthesis and proliferation of bovine mammary epithelial cell. We showed that ATAD3A is localized in mitochondria and the expression of ATAD3A was up-regulated in response to extracellular stimuli such as amino acids and hormones. We observed that ATAD3A positively regulated milk protein, fat, and lactose biosynthesis, and cell proliferation. We further revealed that ATAD3A promoted the expressions of mTOR, SREBP-1c, and Cyclin D1, and triggers mTOR phosphorylation. In summary, our data reveal that ATAD3A regulates the mTOR, SREBP-1c, and Cyclin D1 signaling pathways for milk biosynthesis and cell proliferation. © 2018 International Federation for Cell Biology.

Epithelial stratification and placode invagination are separable functions in early morphogenesis of the molar tooth

PubMed Central

Li, Jingjing; Chatzeli, Lemonia; Panousopoulou, Eleni; Tucker, Abigail S.; Green, Jeremy B. A.

2016-01-01

Ectodermal organs, which include teeth, hair follicles, mammary ducts, and glands such as sweat, mucous and sebaceous glands, are initiated in development as placodes, which are epithelial thickenings that invaginate and bud into the underlying mesenchyme. These placodes are stratified into a basal and several suprabasal layers of cells. The mechanisms driving stratification and invagination are poorly understood. Using the mouse molar tooth as a model for ectodermal organ morphogenesis, we show here that vertical, stratifying cell divisions are enriched in the forming placode and that stratification is cell division dependent. Using inhibitor and gain-of-function experiments, we show that FGF signalling is necessary and sufficient for stratification but not invagination as such. We show that, instead, Shh signalling is necessary for, and promotes, invagination once suprabasal tissue is generated. Shh-dependent suprabasal cell shape suggests convergent migration and intercalation, potentially accounting for post-stratification placode invagination to bud stage. We present a model in which FGF generates suprabasal tissue by asymmetric cell division, while Shh triggers cell rearrangement in this tissue to drive invagination all the way to bud formation. PMID:26755699

A time- and matrix-dependent TGFBR3–JUND–KRT5 regulatory circuit in single breast epithelial cells and basal-like premalignancies

PubMed Central

Wang, Chun-Chao; Bajikar, Sameer S.; Jamal, Leen; Atkins, Kristen A.; Janes, Kevin A.

2014-01-01

Basal-like breast carcinoma is characterized by poor prognosis and high intratumor heterogeneity. In an immortalized basal-like breast epithelial cell line, we identified two anti-correlated gene-expression programs that arise among single extracellular matrix (ECM)-attached cells during organotypic 3D culture. The first contains multiple TGFβ-related genes including TGFBR3, whereas the second contains JUND and the basal-like marker, KRT5. TGFBR3 and JUND interconnect through four negative-feedback loops to form a circuit that exhibits spontaneous damped oscillations in 3D culture. The TGFBR3–JUND circuit appears conserved in some premalignant lesions that heterogeneously express KRT5. The circuit depends on ECM engagement, as detachment causes a rewiring that is triggered by RPS6 dephosphorylation and maintained by juxtacrine tenascin C, which is critical for intraductal colonization of basal-like breast cancer cells in vivo. Intratumor heterogeneity need not stem from partial differentiation and could instead reflect dynamic toggling of cells between expression states that are not cell autonomous. PMID:24658685

Oncogene-like induction of cellular invasion from centrosome amplification

PubMed Central

Godinho, Susana A.; Picone, Remigio; Burute, Mithila; Dagher, Regina; Su, Ying; Leung, Cheuk T.; Polyak, Kornelia; Brugge, Joan S.; Thery, Manuel; Pellman, David

2014-01-01

Centrosome amplification has long been recognized as a feature of human tumors, however its role in tumorigenesis remains unclear1. Centrosome amplification is poorly tolerated by non-transformed cells, and, in the absence of selection, extra centrosomes are spontaneously lost2. Thus, the high frequency of centrosome amplification, particularly in more aggressive tumors3, raises the possibility that extra centrosomes could, in some contexts, confer advantageous characteristics that promote tumor progression. Using a three-dimensional model system and other approaches to culture human mammary epithelial cells, we find that centrosome amplification triggers cell invasion. This invasive behavior is similar to that induced by overexpression of the breast cancer oncogene ErbB24 and indeed enhances invasiveness triggered by ErbB2. We show that, through increased centrosomal microtubule nucleation, centrosome amplification increases Rac1 activity, which disrupts normal cell-cell adhesion and promotes invasion. These findings demonstrate that centrosome amplification, a structural alteration of the cytoskeleton, can promote features of malignant transformation. PMID:24739973

[Immunological background and pathomechanisms of food allergies].

PubMed

Schülke, Stefan; Scheurer, Stephan

2016-06-01

Recent advances in immunology have greatly improved our understanding of the pathomechanisms of food allergies. Food allergies are caused and maintained by complex interactions of the innate and adaptive immune system involving antigen-presenting cells (APC), T cells, group 2 innate lymphoid cells (ILC2), epithelial cells (EC) and effectors cells. Additionally, epigenetic factors, the intestinal microbiome and nutritional factors modulating the gastrointestinal lymphatic tissue probably have a significant impact on allergy development. However, why certain individuals develop tolerance while others mount allergic responses, the factors defining the allergenicity of food proteins, as well as the immunological mechanisms triggering allergy development have yet to be analyzed in detail.

Gap Junctional Coupling is Essential for Epithelial Repair in the Avian Cochlea

PubMed Central

Nickel, Regina; Forge, Andrew

2014-01-01

The loss of auditory hair cells triggers repair responses within the population of nonsensory supporting cells. When hair cells are irreversibly lost from the mammalian cochlea, supporting cells expand to fill the resulting lesions in the sensory epithelium, an initial repair process that is dependent on gap junctional intercellular communication (GJIC). In the chicken cochlea (the basilar papilla or BP), dying hair cells are extruded from the epithelium and supporting cells expand to fill the lesions and then replace hair cells via mitotic and/or conversion mechanisms. Here, we investigated the involvement of GJIC in the initial epithelial repair process in the aminoglycoside-damaged BP. Gentamicin-induced hair cell loss was associated with a decrease of chicken connexin43 (cCx43) immunofluorescence, yet cCx30-labeled gap junction plaques remained. Fluorescence recovery after photobleaching experiments confirmed that the GJIC remained robust in gentamicin-damaged explants, but regionally asymmetric coupling was no longer evident. Dye injections in slice preparations from undamaged BP explants identified cell types with characteristic morphologies along the neural-abneural axis, but these were electrophysiologically indistinct. In gentamicin-damaged BP, supporting cells expanded to fill space formerly occupied by hair cells and displayed more variable electrophysiological phenotypes. When GJIC was inhibited during the aminoglycoside damage paradigm, the epithelial repair response halted. Dying hair cells were retained within the sensory epithelium and supporting cells remained unexpanded. These observations suggest that repair of the auditory epithelium shares common mechanisms across vertebrate species and emphasize the importance of functional gap junctions in maintaining a homeostatic environment permissive for subsequent hair cell regeneration. PMID:25429127

MDA-9/Syntenin (SDCBP) modulates small GTPases RhoA and Cdc42 via transforming growth factor β1 to enhance epithelial-mesenchymal transition in breast cancer.

PubMed

Menezes, Mitchell E; Shen, Xue-Ning; Das, Swadesh K; Emdad, Luni; Sarkar, Devanand; Fisher, Paul B

2016-12-06

Epithelial-mesenchymal transition (EMT) is one of the decisive steps regulating cancer invasion and metastasis. However, the molecular mechanisms underlying this transition require further clarification. MDA-9/syntenin (SDCBP) expression is elevated in breast cancer patient samples as well as cultured breast cancer cells. Silencing expression of MDA-9 in mesenchymal metastatic breast cancer cells triggered a change in cell morphology in both 2D- and 3D-cultures to a more epithelial-like phenotype, along with changes in EMT markers, cytoskeletal rearrangement and decreased invasion. Conversely, over expressing MDA-9 in epithelial non-metastatic breast cancer cells instigated a change in morphology to a more mesenchymal phenotype with corresponding changes in EMT markers, cytoskeletal rearrangement and an increase in invasion. We also found that MDA-9 upregulated active levels of known modulators of EMT, the small GTPases RhoA and Cdc42, via TGFβ1. Reintroducing TGFβ1 in MDA-9 silenced cells restored active RhoA and cdc42 levels, modulated cytoskeletal rearrangement and increased invasion. We further determined that MDA-9 interacts with TGFβ1 via its PDZ1 domain. Finally, in vivo studies demonstrated that silencing the expression of MDA-9 resulted in decreased lung metastasis and TGFβ1 re-expression partially restored lung metastases. Our findings provide evidence for the relevance of MDA-9 in mediating EMT in breast cancer and support the potential of MDA-9 as a therapeutic target against metastatic disease.

Sphingolipid regulation of lung epithelial cell mitophagy and necroptosis during cigarette smoke exposure.

PubMed

Mizumura, Kenji; Justice, Matthew J; Schweitzer, Kelly S; Krishnan, Sheila; Bronova, Irina; Berdyshev, Evgeny V; Hubbard, Walter C; Pewzner-Jung, Yael; Futerman, Anthony H; Choi, Augustine M K; Petrache, Irina

2018-04-01

The mechanisms by which lung structural cells survive toxic exposures to cigarette smoke (CS) are not well defined but may involve proper disposal of damaged mitochondria by macro-autophagy (mitophagy), processes that may be influenced by pro-apoptotic ceramide (Cer) or its precursor dihydroceramide (DHC). Human lung epithelial and endothelial cells exposed to CS exhibited mitochondrial damage, signaled by phosphatase and tensin homolog-induced putative kinase 1 (PINK1) phosphorylation, autophagy, and necroptosis. Although cells responded to CS by rapid inhibition of DHC desaturase, which elevated DHC levels, palmitoyl (C16)-Cer also increased in CS-exposed cells. Whereas DHC augmentation triggered autophagy without cell death, the exogenous administration of C16-Cer was sufficient to trigger necroptosis. Inhibition of Cer-generating acid sphingomyelinase reduced both CS-induced PINK1 phosphorylation and necroptosis. When exposed to CS, Pink1-deficient ( Pink1 -/- ) mice, which are protected from airspace enlargement compared with wild-type littermates, had blunted C16-Cer elevations and less lung necroptosis. CS-exposed Pink1 -/- mice also exhibited significantly increased levels of lignoceroyl (C24)-DHC, along with increased expression of Cer synthase 2 ( CerS2), the enzyme responsible for its production. This suggested that a combination of high C24-DHC and low C16-Cer levels might protect against CS-induced necroptosis. Indeed, CerS2 -/- mice, which lack C24-DHC at the expense of increased C16-Cer, were more susceptible to CS, developing airspace enlargement following only 1 month of exposure. These results implicate DHCs, in particular, C24-DHC, as protective against CS toxicity by enhancing autophagy, whereas C16-Cer accumulation contributes to mitochondrial damage and PINK1-mediated necroptosis, which may be amplified by the inhibition of C24-DHC-producing CerS2.-Mizumura, K., Justice, M. J., Schweitzer, K. S., Krishnan, S., Bronova, I., Berdyshev, E. V., Hubbard, W. C., Pewzner-Jung, Y., Futerman, A. H., Choi, A. M. K., Petrache, I. Sphingolipid regulation of lung epithelial cell mitophagy and necroptosis during cigarette smoke exposure.

Cyanidin-3-O-Glucoside Modulates the In Vitro Inflammatory Crosstalk between Intestinal Epithelial and Endothelial Cells.

PubMed

Ferrari, Daniela; Cimino, Francesco; Fratantonio, Deborah; Molonia, Maria Sofia; Bashllari, Romina; Busà, Rossana; Saija, Antonella; Speciale, Antonio

2017-01-01

Intestinal epithelium represents a protective physical barrier and actively contributes to the mucosal immune system. Polarized basolateral intestinal secretion of inflammatory mediators, followed by activation of NF- κ B signaling and inflammatory pathways in endothelial cells, efficiently triggers extravasation of neutrophils from the vasculature, therefore contributing to the development and maintenance of intestinal inflammation. Proper regulation of NF- κ B activation at the epithelial interface is crucial for the maintenance of physiological tissue homeostasis. Many papers reported that anthocyanins, a group of compounds belonging to flavonoids, possess anti-inflammatory effects and modulate NF- κ B activity. In this study, by using a coculture in vitro system, we aimed to evaluate the effects of TNF- α -stimulated intestinal cells on endothelial cells activation, as well as the protective effects of cyanidin-3-glucoside (C3G). In this model, TNF- α induced nuclear translocation of NF- κ B and TNF- α and IL-8 gene expression in Caco-2 cells, whereas C3G pretreatment dose-dependently reduced these effects. Furthermore, TNF- α -stimulated Caco-2 cells induced endothelial cells activation with increased E-selectin and VCAM-1 mRNA, leukocyte adhesion, and NF- κ B levels in HUVECs, which were inhibited by C3G. We demonstrated that selective inhibition of the NF- κ B pathway in epithelial cells represents the main mechanism by which C3G exerts these protective effects. Thus, anthocyanins could contribute to the management of chronic gut inflammatory diseases.

EGFR-dependent TOR-independent endocycles support Drosophila gut epithelial regeneration.

PubMed

Xiang, Jinyi; Bandura, Jennifer; Zhang, Peng; Jin, Yinhua; Reuter, Hanna; Edgar, Bruce A

2017-05-09

Following gut epithelial damage, epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) signalling triggers Drosophila intestinal stem cells to produce enteroblasts (EBs) and enterocytes (ECs) that regenerate the gut. As EBs differentiate into ECs, they become postmitotic, but undergo extensive growth and DNA endoreplication. Here we report that EGFR/RAS/MAPK signalling is required and sufficient to drive damage-induced EB/EC growth. Endoreplication occurs exclusively in EBs and newborn ECs that inherit EGFR and active MAPK from fast-dividing progenitors. Mature ECs lack EGF receptors and are refractory to growth signalling. Genetic tests indicated that stress-dependent EGFR/MAPK promotes gut regeneration via a novel mechanism that operates independently of Insulin/Pi3K/TOR signalling, which is nevertheless required in nonstressed conditions. The E2f1 transcription factor is required for and sufficient to drive EC endoreplication, and Ras/Raf signalling upregulates E2f1 levels posttranscriptionally. We illustrate how distinct signalling mechanisms direct stress-dependent versus homeostatic regeneration, and highlight the importance of postmitotic cell growth in gut epithelial repair.

EGFR-dependent TOR-independent endocycles support Drosophila gut epithelial regeneration

PubMed Central

Xiang, Jinyi; Bandura, Jennifer; Zhang, Peng; Jin, Yinhua; Reuter, Hanna; Edgar, Bruce A.

2017-01-01

Following gut epithelial damage, epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) signalling triggers Drosophila intestinal stem cells to produce enteroblasts (EBs) and enterocytes (ECs) that regenerate the gut. As EBs differentiate into ECs, they become postmitotic, but undergo extensive growth and DNA endoreplication. Here we report that EGFR/RAS/MAPK signalling is required and sufficient to drive damage-induced EB/EC growth. Endoreplication occurs exclusively in EBs and newborn ECs that inherit EGFR and active MAPK from fast-dividing progenitors. Mature ECs lack EGF receptors and are refractory to growth signalling. Genetic tests indicated that stress-dependent EGFR/MAPK promotes gut regeneration via a novel mechanism that operates independently of Insulin/Pi3K/TOR signalling, which is nevertheless required in nonstressed conditions. The E2f1 transcription factor is required for and sufficient to drive EC endoreplication, and Ras/Raf signalling upregulates E2f1 levels posttranscriptionally. We illustrate how distinct signalling mechanisms direct stress-dependent versus homeostatic regeneration, and highlight the importance of postmitotic cell growth in gut epithelial repair. PMID:28485389

Barriers to horizontal cell transformation by extracellular vesicles containing oncogenic H-ras.

PubMed

Lee, Tae Hoon; Chennakrishnaiah, Shilpa; Meehan, Brian; Montermini, Laura; Garnier, Delphine; D'Asti, Esterina; Hou, Wenyang; Magnus, Nathalie; Gayden, Tenzin; Jabado, Nada; Eppert, Kolja; Majewska, Loydie; Rak, Janusz

2016-08-09

Extracellular vesicles (EVs) enable the exit of regulatory, mutant and oncogenic macromolecules (proteins, RNA and DNA) from their parental tumor cells and uptake of this material by unrelated cellular populations. Among the resulting biological effects of interest is the notion that cancer-derived EVs may mediate horizontal transformation of normal cells through transfer of mutant genes, including mutant ras. Here, we report that H-ras-mediated transformation of intestinal epithelial cells (IEC-18) results in the emission of exosome-like EVs containing genomic DNA, HRAS oncoprotein and transcript. However, EV-mediated horizontal transformation of non-transformed cells (epithelial, astrocytic, fibroblastic and endothelial) is transient, limited or absent due to barrier mechanisms that curtail the uptake, retention and function of oncogenic H-ras in recipient cells. Thus, epithelial cells and astrocytes are resistant to EV uptake, unless they undergo malignant transformation. In contrast, primary and immortalized fibroblasts are susceptible to the EV uptake, retention of H-ras DNA and phenotypic transformation, but these effects are transient and fail to produce a permanent tumorigenic conversion of these cells in vitro and in vivo, even after several months of observation. Increased exposure to EVs isolated from H-ras-transformed cancer cells, but not to those from their indolent counterparts, triggers demise of recipient fibroblasts. Uptake of H-ras-containing EVs stimulates but fails to transform primary endothelial cells. Thus, we suggest that intercellular transfer of oncogenes exerts regulatory rather than transforming influence on recipient cells, while cancer cells may often act as preferential EV recipients.

Barriers to horizontal cell transformation by extracellular vesicles containing oncogenic H-ras

PubMed Central

Lee, Tae Hoon; Chennakrishnaiah, Shilpa; Meehan, Brian; Montermini, Laura; Garnier, Delphine; D'Asti, Esterina; Hou, Wenyang; Magnus, Nathalie; Gayden, Tenzin; Jabado, Nada; Eppert, Kolja; Majewska, Loydie; Rak, Janusz

2016-01-01

Extracellular vesicles (EVs) enable the exit of regulatory, mutant and oncogenic macromolecules (proteins, RNA and DNA) from their parental tumor cells and uptake of this material by unrelated cellular populations. Among the resulting biological effects of interest is the notion that cancer-derived EVs may mediate horizontal transformation of normal cells through transfer of mutant genes, including mutant ras. Here, we report that H-ras-mediated transformation of intestinal epithelial cells (IEC-18) results in the emission of exosome-like EVs containing genomic DNA, HRAS oncoprotein and transcript. However, EV-mediated horizontal transformation of non-transformed cells (epithelial, astrocytic, fibroblastic and endothelial) is transient, limited or absent due to barrier mechanisms that curtail the uptake, retention and function of oncogenic H-ras in recipient cells. Thus, epithelial cells and astrocytes are resistant to EV uptake, unless they undergo malignant transformation. In contrast, primary and immortalized fibroblasts are susceptible to the EV uptake, retention of H-ras DNA and phenotypic transformation, but these effects are transient and fail to produce a permanent tumorigenic conversion of these cells in vitro and in vivo, even after several months of observation. Increased exposure to EVs isolated from H-ras-transformed cancer cells, but not to those from their indolent counterparts, triggers demise of recipient fibroblasts. Uptake of H-ras-containing EVs stimulates but fails to transform primary endothelial cells. Thus, we suggest that intercellular transfer of oncogenes exerts regulatory rather than transforming influence on recipient cells, while cancer cells may often act as preferential EV recipients. PMID:27437771

α-blockade, apoptosis, and prostate shrinkage: how are they related?

PubMed

Chłosta, Piotr; Drewa, Tomasz; Kaplan, Steven

2013-01-01

The α1-adrenoreceptor antagonists, such as terazosin and doxazosin, induce prostate programmed cell death (apoptosis) within prostate epithelial and stromal cells in vitro. This treatment should cause prostate volume decrease, However, this has never been observed in clinical conditions. The aim of this paper is to review the disconnect between these two processes. PubMed and DOAJ were searched for papers related to prostate, apoptosis, and stem cell death. The following key words were used: prostate, benign prostate hyperplasia, programmed cell death, apoptosis, cell death, α1-adrenoreceptor antagonist, α-blockade, prostate epithelium, prostate stroma, stem cells, progenitors, and in vitro models. We have shown how discoveries related to stem cells can influence our understanding of α-blockade treatment for BPH patients. Prostate epithelial and mesenchymal compartments have stem (progenitors) and differentiating cells. These compartments are described in relation to experimental in vitro and in vivo settings. Apoptosis is observed within prostate tissue, but this effect has no clinical significance and cannot lead to prostate shrinkage. In part, this is due to stem cells that are responsible for prostate tissue regeneration and are resistant to apoptosis triggered by α1-receptor antagonists.

Prostaglandin E2 promotes intestinal repair through an adaptive cellular response of the epithelium.

PubMed

Miyoshi, Hiroyuki; VanDussen, Kelli L; Malvin, Nicole P; Ryu, Stacy H; Wang, Yi; Sonnek, Naomi M; Lai, Chin-Wen; Stappenbeck, Thaddeus S

2017-01-04

Adaptive cellular responses are often required during wound repair. Following disruption of the intestinal epithelium, wound-associated epithelial (WAE) cells form the initial barrier over the wound. Our goal was to determine the critical factor that promotes WAE cell differentiation. Using an adaptation of our in vitro primary epithelial cell culture system, we found that prostaglandin E2 (PGE 2 ) signaling through one of its receptors, Ptger4, was sufficient to drive a differentiation state morphologically and transcriptionally similar to in vivo WAE cells. WAE cell differentiation was a permanent state and dominant over enterocyte differentiation in plasticity experiments. WAE cell differentiation was triggered by nuclear β-catenin signaling independent of canonical Wnt signaling. Creation of WAE cells via the PGE 2 -Ptger4 pathway was required in vivo, as mice with loss of Ptger4 in the intestinal epithelium did not produce WAE cells and exhibited impaired wound repair. Our results demonstrate a mechanism by which WAE cells are formed by PGE 2 and suggest a process of adaptive cellular reprogramming of the intestinal epithelium that occurs to ensure proper repair to injury. © 2016 The Authors.

The Kraken Wakes: induced EMT as a driver of tumour aggression and poor outcome.

PubMed

Redfern, Andrew D; Spalding, Lisa J; Thompson, Erik W

2018-06-08

Epithelial mesenchymal transition (EMT) describes the shift of cells from an epithelial form to a contact independent, migratory, mesenchymal form. In cancer the change is linked to invasion and metastasis. Tumour conditions, including hypoxia, acidosis and a range of treatments can trigger EMT, which is implicated in the subsequent development of resistance to those same treatments. Consequently, the degree to which EMT occurs may underpin the entire course of tumour progression and treatment response in a patient. In this review we look past the protective effect of EMT against the initial treatment, to the role of the mesenchymal state, once triggered, in promoting disease growth, spread and future treatment insensitivity. In patients a correlation was found between the propensity of a treatment to induce EMT and failure of that treatment to provide a survival benefit, implicating EMT induction in accelerated tumour progression after treatment cessation. Looking to the mechanisms driving this detrimental effect; increased proliferation, suppressed apoptosis, stem cell induction, augmented angiogenesis, enhanced metastatic dissemination, and immune tolerance, can all result from treatment-induced EMT and could worsen outcome. Evidence also suggests EMT induction with earlier therapies attenuates benefits of later treatments. Looking beyond epithelial tumours, de-differentiation also has therapy-attenuating effects and reversal thereof may yield similar rewards. A range of potential therapies are in development that may address the diverse mechanisms and molecular control systems involved in EMT-induced accelerated progression. Considering the broad reaching effects of mesenchymal shift identified, successful deployment of such treatments could substantially improve patient outcomes.

Aberrant Epithelial-Mesenchymal Hedgehog Signaling Characterizes Barrett's Metaplasia

PubMed Central

Wang, David H.; Clemons, Nicholas J.; Miyashita, Tomoharu; Dupuy, Adam J.; Zhang, Wei; Szczepny, Anette; Corcoran-Schwartz, Ian M.; Wilburn, Daniel L.; Montgomery, Elizabeth A.; Wang, Jean S.; Jenkins, Nancy A.; Copeland, Neal A.; Harmon, John W.; Phillips, Wayne A.; Watkins, D. Neil

2010-01-01

Background & Aims The molecular mechanism underlying epithelial metaplasia in Barrett's esophagus remains unknown. Recognizing that Hedgehog signaling is required for early esophageal development, we sought to determine if the Hedgehog pathway is reactivated in Barrett's esophagus, and if genes downstream of the pathway could promote columnar differentiation of esophageal epithelium. Methods Immunohistochemistry, immunofluorescence, and quantitative real-time PCR were used to analyze clinical specimens, human esophageal cell lines, and mouse esophagi. Human esophageal squamous epithelial (HET-1A) and adenocarcinoma (OE33) cells were subjected to acid treatment and used in transfection experiments. Swiss Webster mice were used in a surgical model of bile reflux injury. An in vivo transplant culture system was created using esophageal epithelium from Sonic hedgehog transgenic mice. Results Marked upregulation of Hedgehog ligand expression, which can be induced by acid or bile exposure, occurs frequently in Barrett's epithelium and is associated with stromal expression of the Hedgehog target genes PTCH1 and BMP4. BMP4 signaling induces expression of SOX9, an intestinal crypt transcription factor, which is highly expressed in Barrett's epithelium. We further show that expression of DMBT1, the human homologue of the columnar cell factor Hensin, occurs in Barrett's epithelium and is induced by SOX9. Finally, transgenic expression of Sonic hedgehog in mouse esophageal epithelium induces expression of stromal Bmp4, epithelial Sox9 and columnar cytokeratins. Conclusions Epithelial Hedgehog ligand expression may contribute to the initiation of Barrett's esophagus through induction of stromal BMP4, which triggers reprogramming of esophageal epithelium in favor of a columnar phenotype. PMID:20138038

Intestinal Intraepithelial Lymphocyte-Enterocyte Crosstalk Regulates Production of Bactericidal Angiogenin 4 by Paneth Cells upon Microbial Challenge

PubMed Central

Dalton, Jane E.; Overweg, Karin; Egan, Charlotte E.; Bongaerts, Roy J.; Newton, Darren J.; Cruickshank, Sheena M.; Andrew, Elizabeth M.; Carding, Simon R.

2013-01-01

Antimicrobial proteins influence intestinal microbial ecology and limit proliferation of pathogens, yet the regulation of their expression has only been partially elucidated. Here, we have identified a putative pathway involving epithelial cells and intestinal intraepithelial lymphocytes (iIELs) that leads to antimicrobial protein (AMP) production by Paneth cells. Mice lacking γδ iIELs (TCRδ-/-) express significantly reduced levels of the AMP angiogenin 4 (Ang4). These mice were also unable to up-regulate Ang4 production following oral challenge by Salmonella, leading to higher levels of mucosal invasion compared to their wild type counterparts during the first 2 hours post-challenge. The transfer of γδ iIELs from wild type (WT) mice to TCRδ-/- mice restored Ang4 production and Salmonella invasion levels were reduced to those obtained in WT mice. The ability to restore Ang4 production in TCRδ-/- mice was shown to be restricted to γδ iIELs expressing Vγ7-encoded TCRs. Using a novel intestinal crypt co-culture system we identified a putative pathway of Ang4 production initiated by exposure to Salmonella, intestinal commensals or microbial antigens that induced intestinal epithelial cells to produce cytokines including IL‑23 in a TLR-mediated manner. Exposure of TCR-Vγ7+ γδ iIELs to IL-23 promoted IL‑22 production, which triggered Paneth cells to secrete Ang4. These findings identify a novel role for γδ iIELs in mucosal defence through sensing immediate epithelial cell cytokine responses and influencing AMP production. This in turn can contribute to the maintenance of intestinal microbial homeostasis and epithelial barrier function, and limit pathogen invasion. PMID:24358364

Intestinal intraepithelial lymphocyte-enterocyte crosstalk regulates production of bactericidal angiogenin 4 by Paneth cells upon microbial challenge.

PubMed

Walker, Catherine R; Hautefort, Isabelle; Dalton, Jane E; Overweg, Karin; Egan, Charlotte E; Bongaerts, Roy J; Newton, Darren J; Cruickshank, Sheena M; Andrew, Elizabeth M; Carding, Simon R

2013-01-01

Antimicrobial proteins influence intestinal microbial ecology and limit proliferation of pathogens, yet the regulation of their expression has only been partially elucidated. Here, we have identified a putative pathway involving epithelial cells and intestinal intraepithelial lymphocytes (iIELs) that leads to antimicrobial protein (AMP) production by Paneth cells. Mice lacking γδ iIELs (TCRδ(-/-)) express significantly reduced levels of the AMP angiogenin 4 (Ang4). These mice were also unable to up-regulate Ang4 production following oral challenge by Salmonella, leading to higher levels of mucosal invasion compared to their wild type counterparts during the first 2 hours post-challenge. The transfer of γδ iIELs from wild type (WT) mice to TCRδ(-/-) mice restored Ang4 production and Salmonella invasion levels were reduced to those obtained in WT mice. The ability to restore Ang4 production in TCRδ(-/-) mice was shown to be restricted to γδ iIELs expressing Vγ7-encoded TCRs. Using a novel intestinal crypt co-culture system we identified a putative pathway of Ang4 production initiated by exposure to Salmonella, intestinal commensals or microbial antigens that induced intestinal epithelial cells to produce cytokines including IL‑23 in a TLR-mediated manner. Exposure of TCR-Vγ7(+) γδ iIELs to IL-23 promoted IL‑22 production, which triggered Paneth cells to secrete Ang4. These findings identify a novel role for γδ iIELs in mucosal defence through sensing immediate epithelial cell cytokine responses and influencing AMP production. This in turn can contribute to the maintenance of intestinal microbial homeostasis and epithelial barrier function, and limit pathogen invasion.

Epigenetic up-regulation of ribosome biogenesis and more aggressive phenotype triggered by the lack of the histone demethylase JHDM1B in mammary epithelial cells

PubMed Central

Galbiati, Alice; Penzo, Marianna; Bacalini, Maria Giulia; Onofrillo, Carmine; Guerrieri, Ania Naila; Garagnani, Paolo; Franceschi, Claudio; Treré, Davide; Montanaro, Lorenzo

2017-01-01

The alterations of ribosome biogenesis and protein synthesis play a direct role in the development of tumors. The accessibility and transcription of ribosomal genes is controlled at several levels, with their epigenetic regulation being one of the most important. Here we explored the JmjC domain-containing histone demethylase 1B (JHDM1B) function in the epigenetic control of rDNA transcription. Since JHDM1B is a negative regulator of gene transcription, we focused on the effects induced by JHDM1B knock-down (KD). We studied the consequences of stable inducible JHDM1B silencing in cell lines derived from transformed and untransformed mammary epithelial cells. In these cellular models, prolonged JHDM1B downregulation triggered a surge of 45S pre-rRNA transcription and processing, associated with a re-modulation of the H3K36me2 levels at rDNA loci and with changes in DNA methylation of specific CpG sites in rDNA genes. We also found that after JHDM1B KD, cells showed a higher ribosome content: which were engaged in mRNA translation. JHDM1B KD and the consequent stimulation of ribosomes biogenesis conferred more aggressive features to the tested cellular models, which acquired a greater clonogenic, staminal and invasive potential. Taken together, these data indicate that the reduction of JHDM1B leads to a more aggressive cellular phenotype in mammary gland cells, by virtue of its negative regulatory activity on ribosome biogenesis. PMID:28415746

Epigenetic up-regulation of ribosome biogenesis and more aggressive phenotype triggered by the lack of the histone demethylase JHDM1B in mammary epithelial cells.

PubMed

Galbiati, Alice; Penzo, Marianna; Bacalini, Maria Giulia; Onofrillo, Carmine; Guerrieri, Ania Naila; Garagnani, Paolo; Franceschi, Claudio; Treré, Davide; Montanaro, Lorenzo

2017-06-06

The alterations of ribosome biogenesis and protein synthesis play a direct role in the development of tumors. The accessibility and transcription of ribosomal genes is controlled at several levels, with their epigenetic regulation being one of the most important. Here we explored the JmjC domain-containing histone demethylase 1B (JHDM1B) function in the epigenetic control of rDNA transcription. Since JHDM1B is a negative regulator of gene transcription, we focused on the effects induced by JHDM1B knock-down (KD). We studied the consequences of stable inducible JHDM1B silencing in cell lines derived from transformed and untransformed mammary epithelial cells. In these cellular models, prolonged JHDM1B downregulation triggered a surge of 45S pre-rRNA transcription and processing, associated with a re-modulation of the H3K36me2 levels at rDNA loci and with changes in DNA methylation of specific CpG sites in rDNA genes. We also found that after JHDM1B KD, cells showed a higher ribosome content: which were engaged in mRNA translation. JHDM1B KD and the consequent stimulation of ribosomes biogenesis conferred more aggressive features to the tested cellular models, which acquired a greater clonogenic, staminal and invasive potential. Taken together, these data indicate that the reduction of JHDM1B leads to a more aggressive cellular phenotype in mammary gland cells, by virtue of its negative regulatory activity on ribosome biogenesis.

Bcl-2 protects tubular epithelial cells from ischemia reperfusion injury by inhibiting apoptosis.

PubMed

Suzuki, Chigure; Isaka, Yoshitaka; Shimizu, Shigeomi; Tsujimoto, Yoshihide; Takabatake, Yoshitsugu; Ito, Takahito; Takahara, Shiro; Imai, Enyu

2008-01-01

Ischemia followed by reperfusion leads to severe organ injury and dysfunction. Inflammation is considered to be the most important cause of graft dysfunction in kidney transplantation subjected to ischemia. The mechanism that triggers inflammation and renal injury after ischemia remains to be elucidated; however, cellular stress may induce apoptosis during the first hours and days after transplantation, which might play a crucial role in early graft dysfunction. Bcl-2 is known to inhibit apoptosis induced by the etiological factors promoting ischemia and reperfusion injury. Accordingly, we hypothesized that an augmentation of the antiapoptotic factor Bcl-2 may thus protect tubular epithelial cells by inhibiting apoptosis, thereby ameliorating the subsequent tubulointerstitial injury. We examined the effects of Bcl-2 overexpression on ischemia-reperfusion (I/R) injury using Bcl-2 transgenic mice (Bcl-2 TG) and their wild-type littermates (WT). To investigate the effects of I/R injury, the left renal artery and vein were clamped for 45 min, followed by reperfusion for 0-96 h. Bcl-2 TG exhibited decreased active caspase protein in the tubular cells, which led to a reduction in TUNEL-positive apoptotic cells. Consequently, interstitial fibrosis and phenotypic changes were ameliorated in Bcl-2 TG. In conclusion, Bcl-2 augmentation protected renal tubular epithelial cells from I/R, and subsequent interstitial injury by inhibiting tubular apoptosis.

Host response to bovine respiratory pathogens.

PubMed

Czuprynski, Charles J

2009-12-01

Bovine respiratory disease (BRD) involves complex interactions amongst viral and bacterial pathogens that can lead to intense pulmonary inflammation (fibrinous pleuropneumonia). Viral infection greatly increases the susceptibility of cattle to secondary infection of the lung with bacterial pathogens like Mannheimia haemolytica and Histophilus somni. The underlying reason for this viral/bacterial synergism, and the manner in which cattle respond to the virulence strategies of the bacterial pathogens, is incompletely understood. Bovine herpesvirus type 1 (BHV-1) infection of bronchial epithelial cells in vitro enhances the binding of M. haemolytica and triggers release of inflammatory mediators that attract and enhance binding of neutrophils. An exotoxin (leukotoxin) released from M. haemolytica further stimulates release of inflammatory mediators and causes leukocyte death. Cattle infected with H. somni frequently display vasculitis. Exposure of bovine endothelial cells to H. somnii or its lipooligosaccharide (LOS) increases endothelium permeability, and makes the surface of the endothelial cells pro-coagulant. These processes are amplified in the presence of platelets. The above findings demonstrate that bovine respiratory pathogens (BHV-1, M. haemolytica and H. somni) interact with leukocytes and other cells (epithelial and endothelial cells) leading to the inflammation that characterizes BRD.

Persistent Transmissible Gastroenteritis Virus Infection Enhances Enterotoxigenic Escherichia coli K88 Adhesion by Promoting Epithelial-Mesenchymal Transition in Intestinal Epithelial Cells.

PubMed

Xia, Lu; Dai, Lei; Yu, Qinghua; Yang, Qian

2017-11-01

Transmissible gastroenteritis virus (TGEV) is a coronavirus characterized by diarrhea and high morbidity rates, and the mortality rate is 100% in piglets less than 2 weeks old. Pigs infected with TGEV often suffer secondary infection by other pathogens, which aggravates the severity of diarrhea, but the mechanisms remain unknown. Here, we hypothesized that persistent TGEV infection stimulates the epithelial-mesenchymal transition (EMT), and thus enterotoxigenic Escherichia coli (ETEC) can more easily adhere to generating cells. Intestinal epithelial cells are the primary targets of TGEV and ETEC infections. We found that TGEV can persistently infect porcine intestinal columnar epithelial cells (IPEC-J2) and cause EMT, consistent with multiple changes in key cell characteristics. Infected cells display fibroblast-like shapes; exhibit increases in levels of mesenchymal markers with a corresponding loss of epithelial markers; have enhanced expression levels of interleukin-1β (IL-1β), IL-6, IL-8, transforming growth factor β (TGF-β), and tumor necrosis factor alpha (TNF-α) mRNAs; and demonstrate increases in migratory and invasive behaviors. Additional experiments showed that the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) signaling pathways via TGF-β is critical for the TGEV-mediated EMT process. Cellular uptake is also modified in cells that have undergone EMT. TGEV-infected cells have higher levels of integrin α5 and fibronectin and exhibit enhanced ETEC K88 adhesion. Reversal of EMT reduces ETEC K88 adhesion and inhibits the expression of integrin α5 and fibronectin. Overall, these results suggest that TGEV infection induces EMT in IPEC-J2 cells, increasing the adhesion of ETEC K88 in the intestine and facilitating dual infection. IMPORTANCE Transmissible gastroenteritis virus (TGEV) causes pig diarrhea and is often followed by secondary infection by other pathogens. In this study, we showed that persistent TGEV infection induces an EMT in porcine intestinal columnar epithelial cells (IPEC-J2) and enhances the adhesion of the secondary pathogen ETEC K88. Additional experiments suggest that integrin α5 and fibronectin play an important role in TGEV-enhanced ETEC K88 adhesion. Reversal of EMT reduces the expression of integrin α5 and fibronectin and also reduces ETEC K88 adhesion. We conclude that TGEV infection triggers EMT and facilitates dual infection. Our results provide new insights into secondary infection and suggest that targeted anti-EMT therapy may have implications for the prevention and treatment of secondary infection. Copyright © 2017 American Society for Microbiology.

The A2B Adenosine Receptor Modulates the Epithelial– Mesenchymal Transition through the Balance of cAMP/PKA and MAPK/ERK Pathway Activation in Human Epithelial Lung Cells

PubMed Central

Giacomelli, Chiara; Daniele, Simona; Romei, Chiara; Tavanti, Laura; Neri, Tommaso; Piano, Ilaria; Celi, Alessandro; Martini, Claudia; Trincavelli, Maria L.

2018-01-01

The epithelial-mesenchymal transition (EMT) is a complex process in which cell phenotype switches from the epithelial to mesenchymal one. The deregulations of this process have been related with the occurrence of different diseases such as lung cancer and fibrosis. In the last decade, several efforts have been devoted in understanding the mechanisms that trigger and sustain this transition process. Adenosine is a purinergic signaling molecule that has been involved in the onset and progression of chronic lung diseases and cancer through the A2B adenosine receptor subtype activation, too. However, the relationship between A2BAR and EMT has not been investigated, yet. Herein, the A2BAR characterization was carried out in human epithelial lung cells. Moreover, the effects of receptor activation on EMT were investigated in the absence and presence of transforming growth factor-beta (TGF-β1), which has been known to promote the transition. The A2BAR activation alone decreased and increased the expression of epithelial markers (E-cadherin) and the mesenchymal one (Vimentin, N-cadherin), respectively, nevertheless a complete EMT was not observed. Surprisingly, the receptor activation counteracted the EMT induced by TGF-β1. Several intracellular pathways regulate the EMT: high levels of cAMP and ERK1/2 phosphorylation has been demonstrated to counteract and promote the transition, respectively. The A2BAR stimulation was able to modulated these two pathways, cAMP/PKA and MAPK/ERK, shifting the fine balance toward activation or inhibition of EMT. In fact, using a selective PKA inhibitor, which blocks the cAMP pathway, the A2BAR-mediated EMT promotion were exacerbated, and conversely the selective inhibition of MAPK/ERK counteracted the receptor-induced transition. These results highlighted the A2BAR as one of the receptors involved in the modulation of EMT process. Nevertheless, its activation is not enough to trigger a complete transition, its ability to affect different intracellular pathways could represent a mechanism at the basis of EMT maintenance/inhibition based on the extracellular microenvironment. Despite further investigations are needed, herein for the first time the A2BAR has been related to the EMT process, and therefore to the different EMT-related pathologies. PMID:29445342

Precommitment low-level Neurog3 expression defines a long-lived mitotic endocrine-biased progenitor pool that drives production of endocrine-committed cells

PubMed Central

Bechard, Matthew E.; Bankaitis, Eric D.; Hipkens, Susan B.; Ustione, Alessandro; Piston, David W.; Yang, Yu-Ping; Magnuson, Mark A.; Wright, Christopher V.E.

2016-01-01

The current model for endocrine cell specification in the pancreas invokes high-level production of the transcription factor Neurogenin 3 (Neurog3) in Sox9+ bipotent epithelial cells as the trigger for endocrine commitment, cell cycle exit, and rapid delamination toward proto-islet clusters. This model posits a transient Neurog3 expression state and short epithelial residence period. We show, however, that a Neurog3TA.LO cell population, defined as Neurog3 transcriptionally active and Sox9+ and often containing nonimmunodetectable Neurog3 protein, has a relatively high mitotic index and prolonged epithelial residency. We propose that this endocrine-biased mitotic progenitor state is functionally separated from a pro-ductal pool and endows them with long-term capacity to make endocrine fate-directed progeny. A novel BAC transgenic Neurog3 reporter detected two types of mitotic behavior in Sox9+ Neurog3TA.LO progenitors, associated with progenitor pool maintenance or derivation of endocrine-committed Neurog3HI cells, respectively. Moreover, limiting Neurog3 expression dramatically increased the proportional representation of Sox9+ Neurog3TA.LO progenitors, with a doubling of its mitotic index relative to normal Neurog3 expression, suggesting that low Neurog3 expression is a defining feature of this cycling endocrine-biased state. We propose that Sox9+ Neurog3TA.LO endocrine-biased progenitors feed production of Neurog3HI endocrine-committed cells during pancreas organogenesis. PMID:27585590

Histamine prevents radiation-induced mesenchymal changes in breast cancer cells.

PubMed

Galarza, Tamara E; Mohamad, Nora A; Táquez Delgado, Mónica A; Vedoya, Guadalupe M; Crescenti, Ernesto J; Bergoc, Rosa M; Martín, Gabriela A; Cricco, Graciela P

2016-09-01

Radiotherapy is a prime option for treatment of solid tumors including breast cancer though side effects are usually present. Experimental evidence shows an increase in invasiveness of several neoplastic cell types through conventional tumor irradiation. The induction of epithelial to mesenchymal transition is proposed as an underlying cause of metastasis triggered by gamma irradiation. Experiments were conducted to investigate the role of histamine on the ionizing radiation-induced epithelial to mesenchymal transition events in breast cancer cells with different invasive phenotype. We also evaluated the potential involvement of Src phosphorylation in the migratory capability of irradiated cells upon histamine treatment. MCF-7 and MDA-MB-231 mammary tumor cells were exposed to a single dose of 2Gy of gamma radiation and five days after irradiation mesenchymal-like phenotypic changes were observed by optical microscope. The expression and subcellular localization of E-cadherin, β-catenin, vimentin and Slug were determined by immunoblot and indirect immunofluorescence. There was a decrease in the epithelial marker E-cadherin expression and an increase in the mesenchymal marker vimentin after irradiation. E-cadherin and β-catenin were mainly localized in cytoplasm. Slug positive nuclei, matrix metalloproteinase-2 activity and cell migration and invasion were significantly increased. In addition, a significant enhancement in Src phosphorylation/activation could be determined by immunoblot in irradiated cells. MCF-7 and MDA-MB-231 cells also received 1 or 20μM histamine during 24h previous to be irradiated. Notably, pre-treatment of breast cancer cells with 20μM histamine prevented the mesenchymal changes induced by ionizing radiation and also reduced the migratory behavior of irradiated cells decreasing phospho-Src levels. Collectively, our results suggest that histamine may block events related to epithelial to mesenchymal transition in irradiated mammary cancer cells and open a perspective for the potential use of histamine to improve radiotherapy efficacy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Quantitative analysis of injury-induced anterior subcapsular cataract in the mouse: a model of lens epithelial cells proliferation and epithelial-mesenchymal transition.

PubMed

Xiao, Wei; Chen, Xiaoyun; Li, Weihua; Ye, Shaobi; Wang, Wencong; Luo, Lixia; Liu, Yizhi

2015-02-10

The mouse lens capsular injury model has been widely used in investigating the mechanisms of anterior subcapsular cataract (ASC) and posterior capsule opacification (PCO), and evaluating the efficacy of antifibrotic compounds. Nevertheless, there is no available protocol to quantitatively assess the treatment outcomes. Our aim is to describe a new method that can successfully quantify the wound and epithelial-mesenchymal transition (EMT) markers expression in vivo. In this model, lens anterior capsule was punctured with a hypodermic needle, which triggered lens epithelial cells (LECs) proliferation and EMT rapidly. Immunofluorescent staining of injured lens anterior capsule whole-mounts revealed the formation of ASC and high expression of EMT markers in the subcapsular plaques. A series of sectional images of lens capsule were acquired from laser scanning confocal microscopy (LSCM) three-dimensional (3D) scanning. Using LSCM Image Browser software, we can not only obtain high resolution stereo images to present the spatial structures of ASC, but also quantify the subcapsular plaques and EMT markers distribution successfully. Moreover, we also demonstrated that histone deacetylases (HDACs) inhibitor TSA significantly prevented injury-induced ASC using this method. Therefore, the present research provides a useful tool to study ASC and PCO biology as well as the efficacy of new therapies.

Mechanistic insights into the impact of Cold Atmospheric Pressure Plasma on human epithelial cell lines

NASA Astrophysics Data System (ADS)

Dezest, Marlène; Chavatte, Laurent; Bourdens, Marion; Quinton, Damien; Camus, Mylène; Garrigues, Luc; Descargues, Pascal; Arbault, Stéphane; Burlet-Schiltz, Odile; Casteilla, Louis; Clément, Franck; Planat, Valérie; Bulteau, Anne-Laure

2017-01-01

Compelling evidence suggests that Cold Atmospheric Pressure Plasma (CAPP) has potential as a new cancer therapy. However, knowledge about cellular signaling events and toxicity subsequent to plasma treatment is still poorly documented. The aim of this study was to focus on the interaction between 3 different types of plasma (He, He-O2, He-N2) and human epithelial cell lines to gain better insight into plasma-cell interaction. We provide evidence that reactive oxygen and nitrogen species (RONS) are inducing cell death by apoptosis and that the proteasome, a major intracellular proteolytic system which is important for tumor cell growth and survival, is a target of (He or He-N2) CAPP. However, RONS are not the only actors involved in cell death; electric field and charged particles could play a significant role especially for He-O2 CAPP. By differential label-free quantitative proteomic analysis we found that CAPP triggers antioxidant and cellular defense but is also affecting extracellular matrix in keratinocytes. Moreover, we found that malignant cells are more resistant to CAPP treatment than normal cells. Taken together, our findings provide insight into potential mechanisms of CAPP-induced proteasome inactivation and the cellular consequences of these events.

Blue light effect on retinal pigment epithelial cells by display devices.

PubMed

Moon, Jiyoung; Yun, Jieun; Yoon, Yeo Dae; Park, Sang-Il; Seo, Young-Jun; Park, Won-Sang; Chu, Hye Yong; Park, Keun Hong; Lee, Myung Yeol; Lee, Chang Woo; Oh, Soo Jin; Kwak, Young-Shin; Jang, Young Pyo; Kang, Jong Soon

2017-05-22

Blue light has high photochemical energy and induces cell apoptosis in retinal pigment epithelial cells. Due to its phototoxicity, retinal hazard by blue light stimulation has been well demonstrated using high intensity light sources. However, it has not been studied whether blue light in the displays, emitting low intensity light, such as those used in today's smartphones, monitors, and TVs, also causes apoptosis in retinal pigment epithelial cells. We attempted to examine the blue light effect on human adult retinal epithelial cells using display devices with different blue light wavelength ranges, the peaks of which specifically appear at 449 nm, 458 nm, and 470 nm. When blue light was illuminated on A2E-loaded ARPE-19 cells using these displays, the display with a blue light peak at a shorter wavelength resulted in an increased production of reactive oxygen species (ROS). Moreover, the reduction of cell viability and induction of caspase-3/7 activity were more evident in A2E-loaded ARPE-19 cells after illumination by the display with a blue light peak at a shorter wavelength, especially at 449 nm. Additionally, white light was tested to examine the effect of blue light in a mixed color illumination with red and green lights. Consistent with the results obtained using only blue light, white light illuminated by display devices with a blue light peak at a shorter wavelength also triggered increased cell death and apoptosis compared to that illuminated by display devices with a blue light peak at longer wavelength. These results show that even at the low intensity utilized in the display devices, blue light can induce ROS production and apoptosis in retinal cells. Our results also suggest that the blue light hazard of display devices might be highly reduced if the display devices contain less short wavelength blue light.

Shigella IpaH0722 E3 Ubiquitin Ligase Effector Targets TRAF2 to Inhibit PKC–NF-κB Activity in Invaded Epithelial Cells

PubMed Central

Ashida, Hiroshi; Nakano, Hiroyasu; Sasakawa, Chihiro

2013-01-01

NF-κB plays a central role in modulating innate immune responses to bacterial infections. Therefore, many bacterial pathogens deploy multiple mechanisms to counteract NF-κB activation. The invasion of and subsequent replication of Shigella within epithelial cells is recognized by various pathogen recognition receptors as pathogen-associated molecular patterns. These receptors trigger innate defense mechanisms via the activation of the NF-κB signaling pathway. Here, we show the inhibition of the NF-κB activation by the delivery of the IpaH E3 ubiquitin ligase family member IpaH0722 using Shigella's type III secretion system. IpaH0722 dampens the acute inflammatory response by preferentially inhibiting the PKC-mediated activation of NF-κB by ubiquitinating TRAF2, a molecule downstream of PKC, and by promoting its proteasome-dependent degradation. PMID:23754945

Development, validation and implementation of an in vitro model for the study of metabolic and immune function in normal and inflamed human colonic epithelium.

PubMed

Pedersen, Gitte

2015-01-01

Ulcerative colitis (UC) and Crohn's disease (CD), collectively referred to as inflammatory bowel disease (IBD), are chronic immune disorders affecting the gastrointestinal tract. The aetiology of IBD remains an enigma, but increasing evidence suggests that the development of IBD may be triggered by a disturbance in the balance between gut commensal bacteria and host response in the intestinal mucosa. It is now known that epithelial cells have the capacity to secrete and respond to a range of immunological mediators and this suggests that these cells play a prominent role in the pathogenesis of IBD. Current knowledge about the intestinal epithelium has mainly been obtained using models based on animal cells, transformed human intestinal cell lines and isolated cells from resected colonic bowel segments. Species difference, malignant origin and confounders related to surgery, obviously make these cell models however less applicable for patophysiological studies. Consequently, there was a clear need for models of representative intestinal epithelial cells that would allow functional and dynamic studies of the differentiated human colonic epithelium in vitro. The primary purpose of this thesis was to explore and validate the optimal conditions for establishing a model based on short-term cultures of human colonic epithelial cells obtained from endoscopical biopsies. The cell cultures were accordingly used to describe the interplay between proinflammatory cytokines and colonic epithelium, with focus on alterations in viability, butyrate metabolism and secretion of a chemokine and metalloproteinases (MMP). Finally, the model was used to characterize expression and activation of receptors like toll like receptor (TLR)9 and peroxisome activated proliferators (PPAR)- known to be important players in regulation of innate and adaptive immune responses in human colonic epithelium. The results showed that it is possible to establish short-term cultures of representative, viable human colonic epithelial cells from endoscopic mucosal biopsies of patients with IBD. Short-time isolation by EGTA/EDTA from colonic biopsies allowed establishment of small scale cultures of epithelial cells which were viable and metabolic active for up to 48 hours in vitro. The cell model preserved important cellular metabolic and immunological functions of the human colonic epithelium, including the ability to oxidate butyrate, detoxificate phenolic compounds and secrete the chemokine interleukin (IL)-8 in vitro. Tumour necrosis factor (TNF)-α and interferon (IFN)-γ are pro-inflammatory cytokines, which are present in increased amounts in inflamed colonic mucosa. The precise mechanisms of cytokine-mediated mucosal injury are unknown, but one might be that TNF-α and IFN-γ directly impair epithelial cell function similar to effects seen on distinct target cells in other autoimmune diseases. Using the model, both cytokines were found directly to impair the viability of colonic epithelial cells and to induce secretion of IL-8 in vitro. Interestingly, the cells from inflamed IBD mucosa were less sensitive to cytokine-induced damage, which suggests that an intrinsic defense mechanism is triggered in these cells, perhaps as a result of exposure to toxic luminal factors or high local cytokine levels in vivo. TNF-α and IFN-γ may also be involved in regulation of intestinal inflammation through stimulation of MMP expression and proteolytic activity. We found that colonic epithelial cells express a range of MMPs and moreover that expression of distinct MMPs is increased in cells from inflamed IBD mucosa. Using a functional peptide cleavage assay it was shown that epithelial cells secreted proteolytic active enzymes and that the functional MMP activity was increased in inflamed IBD mucosa. This suggests that colonic epithelial cells, like myofibroblasts and immune cells, may contribute to local intestinal mucosal damage, through secretion of active MMPs. Disturbance of recognition and discrimination of potentially harmful pathogens from commensals in the intestinal mucosa have increasingly been implicated in the pathogenesis of IBD. Our results revealed that colonic epithelial cells express TLR9, a key pattern recognition receptor. Interestingly, the differentiated epithelial cells, which have been exposed to the luminal bacterial flora in vivo, were unresponsive to TLR9 ligand stimulation, contrasting findings in the epithelial cell line HT-29 that is cultured continuously in bacteria free environment. These findings suggest, theoretically, that colonic epithelium may regulate immune responses to microbial antigens including commensal bacterial DNA through modulation of the TLR9 pathway. Currently, the results are in line with the emerging view, that the epithelium represents an important frontline cellular component of the innate immune system in the gut. PPARγ is a nuclear receptor involved in the regulation of lipid and carbonhydrate metabolism. Recent studies in rodent colitis models suggest that PPARγ also is involved in modulation of inflammatory processes in the colon. Using the model, we characterise expression and activity of PPARs in human colonic epithelium and, additionally, evaluated the functional significance of a possible imbalanced PPARγ regulation in relation to inflammation. Our experiments showed that colonic epithelial cells express PPARγ and furthermore that PPARγ signalling was impaired in inflamed UC epithelium. It was possible to restore PPARγ signalling in the cell cultures by stimulation with rosiglitazone (a synthetic PPARγ ligand) in vitro. Hence, these experiments prompted us to design a small controlled, clinical study exploring the possible stimulatory effects of rosiglitazone (a PPAR ligand) in vivo. Interestingly, it was found that topical application of rosiglitazone in patients with active distal UC reduced clinical activity and mucosal inflammation similar to the effects measured in patients treated with mesalazine enemas. Moreover, rectal application of rosiglitazone induced PPARγ signalling in the epithelium in vivo, supporting the view that activation of PPARγ may be a new potential therapeutic target in the treatment of UC. Overall, the in vitro model of representative human colonic epithelial cells has shown to be a useful technique for detailed studies of metabolic and immunological functions that are important for homeostasis of the colonic epithelium. Currently, the findings support the view that intestinal epithelial cells actively participate in immunological processes in the colonic mucosa. Additionally, the model seems to be applicable for generating and evaluating new therapeutic approaches from laboratory bench to bed line as illustrated by the PPARγ study. It is therefore probable, that studies in models of representative colonic epithelial cells, as the one described here, could contribute with important knowledge about the pathogenesis of human inflammatory colonic diseases also in the future.

Involvement of reactive oxygen species/c-Jun NH{sub 2}-terminal kinase pathway in kotomolide A induces apoptosis in human breast cancer cells

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

Kuo, P.-L.; Chen, C.-Y.; Tzeng, T.-F.

2008-06-01

The anticancer effects of kotomolide A (KTA), a new butanolide constituent isolated from the leaves of Cinnamomum kotoense (Lauraceae), on the two human breast cancer cell lines MCF-7 and MDA-MB-231, were first investigated in our study. KTA exhibited selectively antiproliferative effects in cancer cell lines without showing any toxicity in normal mammary epithelial cells. Treatment of cancer cells with KTA to trigger G2/M phase arrest was associated with increased p21/WAF1 levels and reduced amounts of cyclin A, cyclin B1, cdc2 and cdc25C. KTA induced cancer cell death treatment by triggering mitochondrial and death receptor 5 (DR5) apoptotic pathways, but didmore » not act on the Fas receptor. Exposure of MCF-7 and MDA-MB-231 cells to KTA resulted in cellular glutathione reduction and ROS generation, accompanied by JNK activation and apoptosis. Both antioxidants, NAC and catalase, significantly decreased apoptosis by inhibiting the phosphorylation of JNK and subsequently triggering DR5 cell death pathways. The reduction of JNK expression by siRNA decreased KTA-mediated Bim cleavage, DR5 upregulation and apoptosis. Furthermore, daily KTA i.p. injections in nude mice with MDA-MB-231 s.c. tumors resulted in a 50% decrease of mean tumor volume, compared with vehicle-treated controls. Taken together, the data show that cell death of breast cancer cells in response to KTA is dependent upon ROS generation and JNK activation, triggering intrinsic and extrinsic apoptotic pathways. The ROS/JNK pathway could be a useful target for novel approaches in breast cancer chemotherapy.« less

Myeloperoxidase-induced Genomic DNA-centered Radicals*

PubMed Central

Gomez-Mejiba, Sandra E.; Zhai, Zili; Gimenez, Maria S.; Ashby, Michael T.; Chilakapati, Jaya; Kitchin, Kirk; Mason, Ronald P.; Ramirez, Dario C.

2010-01-01

Myeloperoxidase (MPO) released by activated neutrophils can initiate and promote carcinogenesis. MPO produces hypochlorous acid (HOCl) that oxidizes the genomic DNA in inflammatory cells as well as in surrounding epithelial cells. DNA-centered radicals are early intermediates formed during DNA oxidation. Once formed, DNA-centered radicals decay by mechanisms that are not completely understood, producing a number of oxidation products that are studied as markers of DNA oxidation. In this study we employed the 5,5-dimethyl-1-pyrroline N-oxide-based immuno-spin trapping technique to investigate the MPO-triggered formation of DNA-centered radicals in inflammatory and epithelial cells and to test whether resveratrol blocks HOCl-induced DNA-centered radical formation in these cells. We found that HOCl added exogenously or generated intracellularly by MPO that has been taken up by the cell or by MPO newly synthesized produces DNA-centered radicals inside cells. We also found that resveratrol passed across cell membranes and scavenged HOCl before it reacted with the genomic DNA, thus blocking DNA-centered radical formation. Taken together our results indicate that the formation of DNA-centered radicals by intracellular MPO may be a useful point of therapeutic intervention in inflammation-induced carcinogenesis. PMID:20406811

Glucose, epithelium, and enteric nervous system: dialogue in the dark.

PubMed

Pfannkuche, H; Gäbel, G

2009-06-01

The gastrointestinal epithelium is in close contact with the various components of the chymus, including nutrients, bacteria and toxins. The epithelial barrier has to decide which components are effectively absorbed and which components are extruded. In the small intestine, a nutrient like glucose is mainly absorbed by the sodium linked glucose cotransporter 1 (SGLT1) and the glucose transporter 2 (GLUT2). The expression and activity of both transport proteins is directly linked to the amount of intraluminal glucose. Besides the direct interaction between glucose and the enterocytes, glucose also stimulates different sensory mechanisms within the intestinal wall. The most important types of cells involved in the sensing of intraluminal contents are enteroendocrine cells and neurones of the enteric nervous system. Regarding glucosensing, a distinct type of enteroendocrine cells, the enterochromaffine (EC) cells are involved. Excitation of EC cells by intraluminal glucose results in the release of serotonin (5-HT), which modulates epithelial functions and activates enteric secretomotorneurones. Enteric neurones are not only activated by 5-HT, but also directly by glucose. The activation of different cell types and the subsequent crosstalk between these cells may trigger appropriate absorptive and secretory processes within the intestine.

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

MUC1 enhances invasiveness of pancreatic cancer cells by inducing epithelial to mesenchymal transition

PubMed Central

Roy, Lopamudra Das; Sahraei, Mahnaz; Subramani, Durai B.; Besmer, Dahlia; Nath, Sritama; Tinder, Teresa L.; Bajaj, Ekta; Shanmugam, Kandavel; Lee, Yong Yook; Hwang, Sun IL; Gendler, Sandra J.; Mukherjee, Pinku

2010-01-01

Increased motility and invasiveness of pancreatic cancer cells are associated with epithelial to mesenchymal transition (EMT). Snai1 and Slug are zinc-finger transcription factors that trigger this process by repressing E-cadherin and enhancing vimentin and N-Cadherin protein expression. However, the mechanisms that regulate this activation in pancreatic tumors remain elusive. MUC1, a transmembrane mucin glycoprotein, is associated with the most invasive forms of pancreatic adenocarcinomas (PDA). In this study, we show that over expression of MUC1 in pancreatic cancer cells triggers the molecular process of EMT which translates to increased invasiveness and metastasis. EMT was significantly reduced when Muc1 was genetically deleted in a mouse model of PDA or when all seven tyrosines in the cytoplasmic tail of MUC1 were mutated to phenylalanine (mutated MUC1 CT). Using proteomics, RT-PCR, and Western blotting, we revealed a significant increase in vimentin, Slug and Snail expression with repression of E-Cadherin in MUC1-expressing cells compared to cells expressing the mutated MUC1 CT. In the cells that carried the mutated MUC1 CT, MUC1 failed to co-immunoprecipitate with β-catenin and translocate to the nucleus thereby blocking transcription of the genes associated with EMT and metastasis. Thus, functional tyrosines are critical in stimulating the interactions between MUC1 and β-catenin and their nuclear translocation to initiate the process of EMT. This study signifies the oncogenic role of MUC1 CT and is the first to identify a direct role of the MUC1 in initiating EMT during pancreatic cancer. The data may have implications in future design of MUC1-targeted therapies for pancreatic cancer. PMID:21102519

MUC1 enhances invasiveness of pancreatic cancer cells by inducing epithelial to mesenchymal transition.

PubMed

Roy, L D; Sahraei, M; Subramani, D B; Besmer, D; Nath, S; Tinder, T L; Bajaj, E; Shanmugam, K; Lee, Y Y; Hwang, S I L; Gendler, S J; Mukherjee, P

2011-03-24

Increased motility and invasiveness of pancreatic cancer cells are associated with epithelial to mesenchymal transition (EMT). Snai1 and Slug are zinc-finger transcription factors that trigger this process by repressing E-cadherin and enhancing vimentin and N-cadherin protein expression. However, the mechanisms that regulate this activation in pancreatic tumors remain elusive. MUC1, a transmembrane mucin glycoprotein, is associated with the most invasive forms of pancreatic ductal adenocarcinomas (PDA). In this study, we show that over expression of MUC1 in pancreatic cancer cells triggers the molecular process of EMT, which translates to increased invasiveness and metastasis. EMT was significantly reduced when MUC1 was genetically deleted in a mouse model of PDA or when all seven tyrosines in the cytoplasmic tail of MUC1 were mutated to phenylalanine (mutated MUC1 CT). Using proteomics, RT-PCR and western blotting, we revealed a significant increase in vimentin, Slug and Snail expression with repression of E-Cadherin in MUC1-expressing cells compared with cells expressing the mutated MUC1 CT. In the cells that carried the mutated MUC1 CT, MUC1 failed to co-immunoprecipitate with β-catenin and translocate to the nucleus, thereby blocking transcription of the genes associated with EMT and metastasis. Thus, functional tyrosines are critical in stimulating the interactions between MUC1 and β-catenin and their nuclear translocation to initiate the process of EMT. This study signifies the oncogenic role of MUC1 CT and is the first to identify a direct role of the MUC1 in initiating EMT during pancreatic cancer. The data may have implications in future design of MUC1-targeted therapies for pancreatic cancer.

Respiratory syncytial virus induced type I IFN production by pDC is regulated by RSV-infected airway epithelial cells, RSV-exposed monocytes and virus specific antibodies.

PubMed

Schijf, Marcel A; Lukens, Michael V; Kruijsen, Debby; van Uden, Nathalie O P; Garssen, Johan; Coenjaerts, Frank E J; Van't Land, Belinda; van Bleek, Grada M

2013-01-01

Innate immune responses elicited upon virus exposure are crucial for the effective eradication of viruses, the onset of adaptive immune responses and for establishing proper immune memory. Respiratory syncytial virus (RSV) is responsible for a high disease burden in neonates and immune compromised individuals, causing severe lower respiratory tract infections. During primary infections exuberant innate immune responses may contribute to disease severity. Furthermore, immune memory is often insufficient to protect during RSV re-exposure, which results in frequent symptomatic reinfections. Therefore, identifying the cell types and pattern recognition receptors (PRRs) involved in RSV-specific innate immune responses is necessary to understand incomplete immunity against RSV. We investigated the innate cellular response triggered upon infection of epithelial cells and peripheral blood mononuclear cells. We show that CD14(+) myeloid cells and epithelial cells are the major source of IL-8 and inflammatory cytokines, IL-6 and TNF-α, when exposed to live RSV Three routes of RSV-induced IFN-α production can be distinguished that depend on the cross-talk of different cell types and the presence or absence of virus specific antibodies, whereby pDC are the ultimate source of IFN-α. RSV-specific antibodies facilitate direct TLR7 access into endosomal compartments, while in the absence of antibodies, infection of monocytes or epithelial cells is necessary to provide an early source of type I interferons, required to engage the IFN-α,β receptor (IFNAR)-mediated pathway of IFN-α production by pDC. However, at high pDC density infection with RSV causes IFN-α production without the need for a second party cell. Our study shows that cellular context and immune status are factors affecting innate immune responses to RSV. These issues should therefore be addressed during the process of vaccine development and other interventions for RSV disease.

Respiratory Syncytial Virus Induced Type I IFN Production by pDC Is Regulated by RSV-Infected Airway Epithelial Cells, RSV-Exposed Monocytes and Virus Specific Antibodies

PubMed Central

Schijf, Marcel A.; Lukens, Michael V.; Kruijsen, Debby; van Uden, Nathalie O. P.; Garssen, Johan; Coenjaerts, Frank E. J.; van’t Land, Belinda; van Bleek, Grada M.

2013-01-01

Innate immune responses elicited upon virus exposure are crucial for the effective eradication of viruses, the onset of adaptive immune responses and for establishing proper immune memory. Respiratory syncytial virus (RSV) is responsible for a high disease burden in neonates and immune compromised individuals, causing severe lower respiratory tract infections. During primary infections exuberant innate immune responses may contribute to disease severity. Furthermore, immune memory is often insufficient to protect during RSV re-exposure, which results in frequent symptomatic reinfections. Therefore, identifying the cell types and pattern recognition receptors (PRRs) involved in RSV-specific innate immune responses is necessary to understand incomplete immunity against RSV. We investigated the innate cellular response triggered upon infection of epithelial cells and peripheral blood mononuclear cells. We show that CD14+ myeloid cells and epithelial cells are the major source of IL-8 and inflammatory cytokines, IL-6 and TNF-α, when exposed to live RSV Three routes of RSV-induced IFN-α production can be distinguished that depend on the cross-talk of different cell types and the presence or absence of virus specific antibodies, whereby pDC are the ultimate source of IFN-α. RSV-specific antibodies facilitate direct TLR7 access into endosomal compartments, while in the absence of antibodies, infection of monocytes or epithelial cells is necessary to provide an early source of type I interferons, required to engage the IFN-α,β receptor (IFNAR)-mediated pathway of IFN-α production by pDC. However, at high pDC density infection with RSV causes IFN-α production without the need for a second party cell. Our study shows that cellular context and immune status are factors affecting innate immune responses to RSV. These issues should therefore be addressed during the process of vaccine development and other interventions for RSV disease. PMID:24303065

Restoring conjunctival tolerance by topical nuclear factor-κB inhibitors reduces preservative-facilitated allergic conjunctivitis in mice.

PubMed

Guzmán, Mauricio; Sabbione, Florencia; Gabelloni, María Laura; Vanzulli, Silvia; Trevani, Analía Silvina; Giordano, Mirta Nilda; Galletti, Jeremías Gastón

2014-09-04

To evaluate the role of nuclear factor-κB (NF-κB) activation in eye drop preservative toxicity and the effect of topical NF-κB inhibitors on preservative-facilitated allergic conjunctivitis. Balb/c mice were instilled ovalbumin (OVA) combined with benzalkonium chloride (BAK) and/or NF-κB inhibitors in both eyes. After immunization, T-cell responses and antigen-induced ocular inflammation were evaluated. Nuclear factor-κB activation and associated inflammatory changes also were assessed in murine eyes and in an epithelial cell line after BAK exposure. Benzalkonium chloride promoted allergic inflammation and leukocyte infiltration of the conjunctiva. Topical NF-κB inhibitors blocked the disruptive effect of BAK on conjunctival immunological tolerance and ameliorated subsequent ocular allergic reactions. In line with these findings, BAK induced NF-κB activation and the secretion of IL-6 and granulocyte-monocyte colony-stimulating factor in an epithelial cell line and in the conjunctiva of instilled mice. In addition, BAK favored major histocompatibility complex (MHC) II expression in cultured epithelial cells in an NF-κB-dependent fashion after interaction with T cells. Benzalkonium chloride triggers conjunctival epithelial NF-κB activation, which seems to mediate some of its immune side effects, such as proinflammatory cytokine release and increased MHC II expression. Breakdown of conjunctival tolerance by BAK favors allergic inflammation, and this effect can be prevented in mice by topical NF-κB inhibitors. These results suggest a new pharmacological target for preservative toxicity and highlight the importance of conjunctival tolerance in ocular surface homeostasis. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Variation of HPV Subtypes with Focus on HPV-Infection and Cancer in the Head and Neck Region.

PubMed

Wichmann, Gunnar

The human papillomavirus (HPV) comprises a heterogeneous group of double-strand DNA viruses with variable potential to infect human epithelial cells and trigger neoplastic transformation. Its 8 kb genome encodes proteins required for virus replication and self-organized formation of infectious particles but also for early proteins E6 and E7 able to trigger neoplastic transformation. E6 and E7 of high-risk (HR) HPV subtypes can bind to p53 or release E2F and abrogate replication control. Due to variable amino acid sequence (AAS) in the binding sites of E6 and E7 particular HR-HPV variants within subtypes are essentially heterogeneous in efficacy triggering neoplastic transformation and cancer development. This could explain differences in the clinical course of HPV-driven head and neck cancer.

The cytotoxic effect of oxybuprocaine on human corneal epithelial cells by inducing cell cycle arrest and mitochondria-dependent apoptosis.

PubMed

Fan, W-Y; Wang, D-P; Wen, Q; Fan, T-J

2017-08-01

Oxybuprocaine (OBPC) is a widely used topical anesthetic in eye clinic, and prolonged and repeated usage of OBPC might be cytotoxic to the cornea, especially to the outmost corneal epithelium. In this study, we characterized the cytotoxic effect of OBPC on human corneal epithelial (HCEP) cells and investigated its possible cellular and molecular mechanisms using an in vitro model of non-transfected HCEP cells. Our results showed that OBPC at concentrations ranging from 0.025% to 0.4% had a dose- and time-dependent cytotoxicity to HCEP cells. Moreover, OBPC arrested the cells at S phase and induced apoptosis of these cells by inducing plasma membrane permeability, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation. Furthermore, OBPC could trigger the activation of caspase-2, -3, and -9, downregulate the expression of Bcl-xL, upregulate the expression of Bax along with the cytoplasmic amount of mitochondria-released apoptosis-inducing factor, and disrupt mitochondrial transmembrane potential. Our results suggest that OBPC has a dose- and time-dependent cytotoxicity to HCEP cells by inducing cell cycle arrest and cell apoptosis via a death receptor-mediated mitochondria-dependent proapoptotic pathway, and this novel finding provides new insights into the acute cytotoxicity and its toxic mechanisms of OBPC on HCEP cells.

Commensal Bacteria Modulate Innate Immune Responses of Vaginal Epithelial Cell Multilayer Cultures

PubMed Central

Rose, William A.; McGowin, Chris L.; Spagnuolo, Rae Ann; Eaves-Pyles, Tonyia D.; Popov, Vsevolod L.; Pyles, Richard B.

2012-01-01

The human vaginal microbiome plays a critical but poorly defined role in reproductive health. Vaginal microbiome alterations are associated with increased susceptibility to sexually-transmitted infections (STI) possibly due to related changes in innate defense responses from epithelial cells. Study of the impact of commensal bacteria on the vaginal mucosal surface has been hindered by current vaginal epithelial cell (VEC) culture systems that lack an appropriate interface between the apical surface of stratified squamous epithelium and the air-filled vaginal lumen. Therefore we developed a reproducible multilayer VEC culture system with an apical (luminal) air-interface that supported colonization with selected commensal bacteria. Multilayer VEC developed tight-junctions and other hallmarks of the vaginal mucosa including predictable proinflammatory cytokine secretion following TLR stimulation. Colonization of multilayers by common vaginal commensals including Lactobacillus crispatus, L. jensenii, and L. rhamnosus led to intimate associations with the VEC exclusively on the apical surface. Vaginal commensals did not trigger cytokine secretion but Staphylococcus epidermidis, a skin commensal, was inflammatory. Lactobacilli reduced cytokine secretion in an isolate-specific fashion following TLR stimulation. This tempering of inflammation offers a potential explanation for increased susceptibility to STI in the absence of common commensals and has implications for testing of potential STI preventatives. PMID:22412914

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

PubMed

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

2017-03-01

There are two principal senescence barriers that must be overcome to successfully immortalize primary human epithelial cells in culture, stress-induced senescence, and replicative senescence. The p16 INK4a /retinoblastoma protein (p16/Rb) pathway mediates stress-induced senescence, and is generally upregulated by primary epithelial cells in response to the artificial conditions from tissue culture. Replicative senescence is associated with telomere loss. Following each round of cell division, telomeres progressively shorten. Once telomeres shorten to a critical length, the DNA damage response pathway is activated, and the tumor suppressor p53 pathway triggers replicative senescence. Exogenous expression of telomerase in normal human epithelial cells extends the replicative capacity of cells, and in some cases, immortalizes cells. However reliable immortalization of epithelial cells usually requires telomerase activity coupled with inactivation of the p16/Rb pathway. A lentiviral vector, pLOX-TERT-iresTK (Addgene #12245), containing a CMV promoter upstream of a bicistronic coding cassette that includes loxP sites flanking the catalytic subunit of human telomerase gene (TERT) and herpes simplex virus type-1 thymidine kinase gene (HSV1-tk) was used to transduce normal prostate basal epithelial cells (PrECs) initiated in cell culture from prostate cancer patients undergoing radical prostatectomies. Transduction of early (i.e., <7) passage PrECs with TERT led to successful immortalization. However, attempts to immortalize late (i.e., >7) passage PrECs were unsuccessful. Late passage PrECs, which acquired elevated p16, were unable to overcome the senescence barrier. Immortalized PrECs (TERT-PrECs) retained a normal male karyotype and low p16 expression. Additionally, TERT-PrECs were non-tumorigenic when inoculated into intact male immunodeficient NSG mice. The present studies document that early passage human PrECs have sufficiently low p16 to permit immortalization by TERT expression alone. TERT-PrECs developed using this transduction approach provides an appropriate and experimentally facile model for clarifying the molecular mechanism(s) involved in both immortalization of human PrECs, as well as identifying genetic/epigenetic "drivers" for conversion of these immortalized non-tumorigenic cells into fully lethal prostate cancers. Notably, loxP sites flank the exogenous TERT gene in the TERT-PrECs. Cre recombinase can be used to excise TERT, and resolve whether TERT expression is required for these cells to be fully transformed into lethal cancer. Prostate 77: 374-384, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

STAT6 deficiency ameliorates Graves' disease severity by suppressing thyroid epithelial cell hyperplasia.

PubMed

Jiang, Xuechao; Zha, Bingbing; Liu, Xiaoming; Liu, Ronghua; Liu, Jun; Huang, Enyu; Qian, Tingting; Liu, Jiajing; Wang, Zhiming; Zhang, Dan; Wang, Luman; Chu, Yiwei

2016-12-01

Signal transducer and activator of transcription 6 (STAT6) is involved in epithelial cell growth. However, little is known regarding the STAT6 phosphorylation status in Graves' disease (GD) and its role in thyroid epithelial cells (TECs). In this study, we found that STAT6 phosphorylation (p-STAT6) was significantly increased in TECs from both GD patients and experimental autoimmune Graves' disease mice and that STAT6 deficiency ameliorated GD symptoms. Autocrine IL-4 signalling in TECs activated the phosphorylation of STAT6 via IL-4 R engagement, and the downstream targets of STAT6 were Bcl-xL and cyclin D1. Thus, the IL-4-STAT6-Bcl-xL/cyclin D1 pathway is crucial for TEC hyperplasia, which aggravates GD. More importantly, in vitro and in vivo experiments demonstrated that STAT6 phosphorylation inhibited by AS1517499 decreased TEC hyperplasia, thereby reducing serum T3 and T4 and ameliorating GD. Thus, our study reveals that in addition to the traditional pathogenesis of GD, in which autoantibody TRAb stimulates thyroid-stimulating hormone receptors and consequently produces T3, T4, TRAb could also trigger TECs producing IL-4, and IL-4 then acts in an autocrine manner to activate p-STAT6 signalling and stimulate unrestricted cell growth, thus aggravating GD. These findings suggest that STAT6 inhibitors could be potent therapeutics for treating GD.

STAT6 deficiency ameliorates Graves' disease severity by suppressing thyroid epithelial cell hyperplasia

PubMed Central

Jiang, Xuechao; Zha, Bingbing; Liu, Xiaoming; Liu, Ronghua; Liu, Jun; Huang, Enyu; Qian, Tingting; Liu, Jiajing; Wang, Zhiming; Zhang, Dan; Wang, Luman; Chu, Yiwei

2016-01-01

Signal transducer and activator of transcription 6 (STAT6) is involved in epithelial cell growth. However, little is known regarding the STAT6 phosphorylation status in Graves' disease (GD) and its role in thyroid epithelial cells (TECs). In this study, we found that STAT6 phosphorylation (p-STAT6) was significantly increased in TECs from both GD patients and experimental autoimmune Graves' disease mice and that STAT6 deficiency ameliorated GD symptoms. Autocrine IL-4 signalling in TECs activated the phosphorylation of STAT6 via IL-4 R engagement, and the downstream targets of STAT6 were Bcl-xL and cyclin D1. Thus, the IL-4-STAT6-Bcl-xL/cyclin D1 pathway is crucial for TEC hyperplasia, which aggravates GD. More importantly, in vitro and in vivo experiments demonstrated that STAT6 phosphorylation inhibited by AS1517499 decreased TEC hyperplasia, thereby reducing serum T3 and T4 and ameliorating GD. Thus, our study reveals that in addition to the traditional pathogenesis of GD, in which autoantibody TRAb stimulates thyroid-stimulating hormone receptors and consequently produces T3, T4, TRAb could also trigger TECs producing IL-4, and IL-4 then acts in an autocrine manner to activate p-STAT6 signalling and stimulate unrestricted cell growth, thus aggravating GD. These findings suggest that STAT6 inhibitors could be potent therapeutics for treating GD. PMID:27906181

LOX-1 activation by oxLDL triggers an epithelial mesenchymal transition and promotes tumorigenic potential in prostate cancer cells.

PubMed

González-Chavarría, I; Fernandez, E; Gutierrez, N; González-Horta, E E; Sandoval, F; Cifuentes, P; Castillo, C; Cerro, R; Sanchez, O; Toledo, Jorge R

2018-02-01

Obesity is related to an increased risk of developing prostate cancer with high malignancy stages or metastasis. Recent results demonstrated that LOX-1, a receptor associated with obesity and atherosclerosis, is overexpressed in advanced and metastatic prostate cancer. Furthermore, high levels of oxLDL, the main ligand for LOX-1, have been found in patients with advanced prostate cancer. However, the role of LOX-1 in prostate cancer has not been unraveled completely yet. Here, we show that LOX-1 is overexpressed in prostate cancer cells and its activation by oxLDL promotes an epithelial to mesenchymal transition, through of lowered expression of epithelial markers (E-cadherin and plakoglobin) and an increased expression of mesenchymal markers (vimentin, N-cadherin, snail, slug, MMP-2 and MMP-9). Consequently, LOX-1 activation by oxLDL promotes actin cytoskeleton restructuration and MMP-2 and MMP-9 activity inducing prostate cancer cell invasion and migration. Additionally, LOX-1 increased the tumorigenic potential of prostate cancer cells and its expression was necessary for tumor growth in nude mice. In conclusion, our results suggest that oxLDL/LOX-1 could be ones of mechanisms that explain why obese patients with prostate cancer have an accelerated tumor progression and a greater probability of developing metastasis. Copyright © 2017 Elsevier B.V. All rights reserved.

Epithelial-to-endothelial transition and cancer stem cells: two cornerstones of vasculogenic mimicry in malignant tumors.

PubMed

Sun, Baocun; Zhang, Danfang; Zhao, Nan; Zhao, Xiulan

2017-05-02

Vasculogenic mimicry (VM) is a functional microcirculation pattern in malignant tumors accompanied by endothelium-dependent vessels and mosaic vessels. VM has been identified in more than 15 solid tumor types and is associated with poor differentiation, late clinical stage and poor prognosis. Classic anti-angiogenic agents do not target endothelium-dependent vessels and are not efficacious against tumors exhibiting VM. Further insight into the molecular signaling that triggers and promotes VM formation could improve anti-angiogenic therapeutics. Recent studies have shown that cancer stem cells (CSCs) and epithelium-to-endothelium transition (EET), a subtype of epithelial-to-mesenchymal transition (EMT), accelerate VM formation by stimulating tumor cell plasticity, remodeling the extracellular matrix (ECM) and connecting VM channels with host blood vessels. VM channel-lining cells originate from CSCs due to expression of EMT inducers such as Twist1, which promote EET and ECM remodeling. Hypoxia and high interstitial fluid pressure in the tumor microenvironment induce a specific type of cell death, linearly patterned programmed cell necrosis (LPPCN), which spatially guides VM and endothelium-dependent vessel networks. This review focuses on the roles of CSCs and EET in VM, and on possible novel anti-angiogenic strategies against alternative tumor vascularization.

Flavonoid Fraction of Orange and Bergamot Juices Protect Human Lung Epithelial Cells from Hydrogen Peroxide-Induced Oxidative Stress

PubMed Central

Ferlazzo, Nadia; Visalli, Giuseppa; Smeriglio, Antonella; Cirmi, Santa; Lombardo, Giovanni Enrico; Campiglia, Pietro; Di Pietro, Angela

2015-01-01

It has been reported that oxidant/antioxidant imbalance triggers cell damage that in turn causes a number of lung diseases. Flavonoids are known for their health benefits, and Citrus fruits juices are one of the main food sources of these secondary plant metabolites. The present study was designed to evaluate the effect of the flavonoid fraction of bergamot and orange juices, on H2O2-induced oxidative stress in human lung epithelial A549 cells. First we tested the antioxidant properties of both extracts in cell-free experimental models and then we assayed their capability to prevent the cytotoxic effects induced by H2O2. Our results demonstrated that both Citrus juice extracts reduce the generation of reactive oxygen species and membrane lipid peroxidation, improve mitochondrial functionality, and prevent DNA-oxidative damage in A549 cells incubated with H2O2. Our data indicate that the mix of flavonoids present in both bergamot and orange juices may be of use in preventing oxidative cell injury and pave the way for further research into a novel healthy approach to avoid lung disorders. PMID:26221182

Flavonoid Fraction of Orange and Bergamot Juices Protect Human Lung Epithelial Cells from Hydrogen Peroxide-Induced Oxidative Stress.

PubMed

Ferlazzo, Nadia; Visalli, Giuseppa; Smeriglio, Antonella; Cirmi, Santa; Lombardo, Giovanni Enrico; Campiglia, Pietro; Di Pietro, Angela; Navarra, Michele

2015-01-01

It has been reported that oxidant/antioxidant imbalance triggers cell damage that in turn causes a number of lung diseases. Flavonoids are known for their health benefits, and Citrus fruits juices are one of the main food sources of these secondary plant metabolites. The present study was designed to evaluate the effect of the flavonoid fraction of bergamot and orange juices, on H2O2-induced oxidative stress in human lung epithelial A549 cells. First we tested the antioxidant properties of both extracts in cell-free experimental models and then we assayed their capability to prevent the cytotoxic effects induced by H2O2. Our results demonstrated that both Citrus juice extracts reduce the generation of reactive oxygen species and membrane lipid peroxidation, improve mitochondrial functionality, and prevent DNA-oxidative damage in A549 cells incubated with H2O2. Our data indicate that the mix of flavonoids present in both bergamot and orange juices may be of use in preventing oxidative cell injury and pave the way for further research into a novel healthy approach to avoid lung disorders.

Tangeretin enhances radiosensitivity and inhibits the radiation-induced epithelial-mesenchymal transition of gastric cancer cells.

PubMed

Zhang, Xukui; Zheng, Luming; Sun, Yinggang; Wang, Tianxiao; Wang, Baocheng

2015-07-01

Irradiation has been reported to increase radioresistance and epithelial-mesenchymal transition (EMT) in gastric cancer (GC) cells. The Notch pathway is critically implicated in cancer EMT and radioresistance. In the present study, we investigated the use of a Notch-1 inhibiting compound as a novel therapeutic candidate to regulate radiation-induced EMT in GC cells. According to previous screening, tangeretin, a polymethoxylated flavonoid from citrus fruits was selected as a Notch-1 inhibitor. Tangeretin enhanced the radiosensitivity of GC cells as demonstrated by MTT and colony formation assays. Tangeretin also attenuated radiation-induced EMT, invasion and migration in GC cells, accompanied by a decrease in Notch-1, Jagged1/2, Hey-1 and Hes-1 expressions. Tangeretin triggered the upregulation of miR-410, a tumor-suppressive microRNA. Furthermore, re-expression of miR-410 prevented radiation-induced EMT and cell invasion. An in vivo tumor xenograft model confirmed the antimetastasis effect of tangeretin as we observed in vitro. In nude mice, tumor size was considerably diminished by radiation plus tangeretin co-treatment. Tangeretin almost completely inhibited lung metastasis induced by irradiation. Tangeretin may be a novel antimetastatic agent for radiotherapy.

Human Mut T Homolog 1 (MTH1): a roadblock for the tumor-suppressive effects of oncogenic RAS-induced ROS.

PubMed

Rai, Priyamvada

2012-01-01

Oncogenic RAS-induced reactive oxygen species (ROS) trigger barriers to cell transformation and cancer progression through tumor-suppressive responses such as cellular senescence or cell death. We have recently shown that oncogenic RAS-induced DNA damage and attendant premature senescence can be prevented by overexpressing human MutT Homolog 1 (MTH1), the major mammalian detoxifier of the oxidized DNA precursor, 8-oxo-dGTP. Paradoxically, RAS-induced ROS are also able to participate in tumor progression via transformative processes such as mitogenic signaling, the epithelial-mesenchymal transition (EMT), anoikis inhibition, and PI3K/Akt-mediated survival signaling. Here we provide a preliminary insight into the influence of MTH1 levels on the EMT phenotype and Akt activation in RAS-transformed HMLE breast epithelial cells. Within this context, we will discuss the implications of MTH1 upregulation in oncogenic RAS-sustaining cells as a beneficial adaptive change that inhibits ROS-mediated cell senescence and participates in the maintenance of ROS-associated tumor-promoting mechanisms. Accordingly, targeting MTH1 in RAS-transformed tumor cells will not only induce proliferative defects but also potentially enhance therapeutic cytotoxicity by shifting cellular response away from pro-survival mechanisms.

Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells.

PubMed

Voynow, J A; Young, L R; Wang, Y; Horger, T; Rose, M C; Fischer, B M

1999-05-01

Chronic neutrophil-predominant inflammation and hypersecretion of mucus are common pathophysiological features of cystic fibrosis, chronic bronchitis, and viral- or pollution-triggered asthma. Neutrophils release elastase, a serine protease, that causes increased mucin production and secretion. The molecular mechanisms of elastase-induced mucin production are unknown. We hypothesized that as part of this mechanism, elastase upregulates expression of a major respiratory mucin gene, MUC5AC. A549, a human lung carcinoma cell line that expresses MUC5AC mRNA and protein, and normal human bronchial epithelial cells in an air-liquid interface culture were stimulated with neutrophil elastase. Neutrophil elastase increased MUC5AC mRNA levels in a time-dependent manner in both cell culture systems. Neutrophil elastase treatment also increased MUC5AC protein levels in A549 cells. The mechanism of MUC5AC gene regulation by elastase was determined in A549 cells. The induction of MUC5AC gene expression required serine protease activity; other classes of proteases had no effect on MUC5AC gene expression. Neutrophil elastase increased MUC5AC mRNA levels by enhancing mRNA stability. This is the first report of mucin gene regulation by this mechanism.

Long-term low-dose α-particle enhanced the potential of malignant transformation in human bronchial epithelial cells through MAPK/Akt pathway

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

Liu, Weili; Xiao, Linlin; Dong, Chen

2014-05-09

Highlights: • Multi-exposures of 25 mGy α-ray enhanced cell proliferation, adhesion, and invasion. • MAPK/Akt but not JNK/P66 was positively correlated with cell invasive phenotypes. • LDR of α-irradiation triggers cell malignant transformation through MAPK/Akt. - Abstract: Since the wide usage of ionizing radiation, the cancer risk of low dose radiation (LDR) (<0.1 Gy) has become attractive for a long time. However, most results are derived from epidemiologic studies on atomic-bomb survivors and nuclear accidents surrounding population, and the molecular mechanism of this risk is elusive. To explore the potential of a long-term LDR-induced malignant transformation, human bronchial epithelial cellsmore » Beas-2B were fractionally irradiated with 0.025 Gy α-particles for 8 times in total and then further cultured for 1–2 months. It was found that the cell proliferation, the abilities of adhesion and invasion, and the protein expressions of p-ERK, p-Akt, especially p-P38 were not only increased in the multiply-irradiated cells but also in their offspring 1–2 months after the final exposure, indicating high potentiality of cell malignant transformation. On opposite, the expressions of p-JNK and p-P66 were diminished in the subcultures of irradiated cells and thus may play a role of negative regulation in canceration. When the cells were transferred with p38 siRNA, the LDR-induced enhancements of cell adhesion and invasion were significantly reduced. These findings suggest that long-term LDR of α-particles could enhance the potential of malignant transformation incidence in human bronchial epithelial cells through MAPK/Akt pathway.« less

Mechanisms underlying differential food allergy response to heated egg.

PubMed

Martos, Gustavo; Lopez-Exposito, Ivan; Bencharitiwong, Ramon; Berin, M Cecilia; Nowak-Węgrzyn, Anna

2011-04-01

Egg white proteins are usually subjected to heating, making them edible for the majority of children with egg allergy. We sought to investigate the underlying mechanisms responsible for the reduced allergenicity displayed by heat-treated egg white allergens. C3H/HeJ mice were orally sensitized with ovalbumin (OVA) or ovomucoid and challenged with native or heated proteins to evaluate their allergenicity. Immunoreactivity was assessed by immunoblotting using sera from children with egg allergy. In vitro gastrointestinal digestion of native and heated OVA and ovomucoid was studied by SDS-PAGE and liquid chromatography. Intestinal uptake of intact native and heated OVA and ovomucoid by human intestinal epithelial (Caco-2) cells was investigated. Rat basophil leukemia cells passively sensitized with mouse serum and human basophils passively sensitized with serum from children with egg allergy were used to assess the effector cell activation by heated, digested, and transported OVA and ovomucoid. Heated OVA and ovomucoid did not induce symptoms of anaphylaxis in sensitized mice when administered orally. Heating did not completely destroy IgE-binding capacity of OVA or ovomucoid but enhanced in vitro digestibility of OVA. Digestion of both OVA and ovomucoid diminished mediator release in rat basophil leukemia assay and basophil activation. Heating of allergens prevented transport across human intestinal epithelial cells in a form capable of triggering basophil activation or T-cell activation. Heat treatment reduces allergenicity of OVA and ovomucoid. This is partially a result of the enhanced gastrointestinal digestibility of heated OVA and the inability of heated OVA or ovomucoid to be absorbed in a form capable of triggering basophils. Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5

PubMed Central

Cavarelli, Mariangela; Foglieni, Chiara; Rescigno, Maria; Scarlatti, Gabriella

2013-01-01

The gastrointestinal tract is a principal route of entry and site of persistence of human immunodeficiency virus type 1 (HIV-1). The intestinal mucosa, being rich of cells that are the main target of the virus, represents a primary site of viral replication and CD4+ T-cell depletion. Here, we show both in vitro and ex vivo that HIV-1 of R5 but not X4 phenotype is capable of selectively triggering dendritic cells (DCs) to migrate within 30 min between intestinal epithelial cells to sample virions and transfer infection to target cells. The engagement of the chemokine receptor 5 on DCs and the viral envelope, regardless of the genetic subtype, drive DC migration. Viruses penetrating through transient opening of the tight junctions likely create a paracellular gradient to attract DCs. The formation of junctions with epithelial cells may initiate a haptotactic process of DCs and at the same time favour cell-to-cell viral transmission. Our findings indicate that HIV-1 translocation across the intestinal mucosa occurs through the selective engagement of DCs by R5 viruses, and may guide the design of new prevention strategies. PMID:23606583

R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.

PubMed

Cavarelli, Mariangela; Foglieni, Chiara; Rescigno, Maria; Scarlatti, Gabriella

2013-05-01

The gastrointestinal tract is a principal route of entry and site of persistence of human immunodeficiency virus type 1 (HIV-1). The intestinal mucosa, being rich of cells that are the main target of the virus, represents a primary site of viral replication and CD4(+) T-cell depletion. Here, we show both in vitro and ex vivo that HIV-1 of R5 but not X4 phenotype is capable of selectively triggering dendritic cells (DCs) to migrate within 30 min between intestinal epithelial cells to sample virions and transfer infection to target cells. The engagement of the chemokine receptor 5 on DCs and the viral envelope, regardless of the genetic subtype, drive DC migration. Viruses penetrating through transient opening of the tight junctions likely create a paracellular gradient to attract DCs. The formation of junctions with epithelial cells may initiate a haptotactic process of DCs and at the same time favour cell-to-cell viral transmission. Our findings indicate that HIV-1 translocation across the intestinal mucosa occurs through the selective engagement of DCs by R5 viruses, and may guide the design of new prevention strategies. Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

Galectin-3 Inhibits Galectin-8/Parkin-Mediated Ubiquitination of Group A Streptococcus.

PubMed

Cheng, Yi-Lin; Wu, Yan-Wei; Kuo, Chih-Feng; Lu, Shiou-Ling; Liu, Fu-Tong; Anderson, Robert; Lin, Chiou-Feng; Liu, Yi-Ling; Wang, Wan-Yu; Chen, Ying-Da; Zheng, Po-Xing; Wu, Jiunn-Jong; Lin, Yee-Shin

2017-07-25

Group A streptococcus (GAS) is an important human pathogen that causes a wide variety of cutaneous and systemic infections. Although originally thought to be an extracellular bacterium, numerous studies have demonstrated that GAS can trigger internalization into nonimmune cells to escape from immune surveillance or antibiotic-mediated killing. Epithelial cells possess a defense mechanism involving autophagy-mediated targeting and killing of GAS within lysosome-fused autophagosomes. In endothelial cells, in contrast, we previously showed that autophagy is not sufficient for GAS killing. In the present study, we showed higher galectin-3 (Gal-3) expression and lower Gal-8 expression in endothelial cells than in epithelial cells. The recruitment of Gal-3 to GAS is higher and the recruitment of Gal-8 to GAS is lower in endothelial cells than in epithelial cells. We further showed that Gal-3 promotes GAS replication and diminishes the recruitment of Gal-8 and ubiquitin, the latter of which is a critical protein for autophagy sequestration. After knockdown of Gal-3 in endothelial cells, the colocalization of Gal-8, parkin, and ubiquitin-decorated GAS is significantly increased, as is the interaction of Gal-8 and parkin, an E3 ligase. Furthermore, inhibition of Gal-8 in epithelial cells attenuates recruitment of parkin; both Gal-8 and parkin contribute to ubiquitin recruitment and GAS elimination. Animal studies confirmed that Gal-3-knockout mice develop less-severe skin damage and that GAS replication can be detected only in the air pouch and not in organs and endothelial cells. These results demonstrate that Gal-3 inhibits ubiquitin recruitment by blocking Gal-8 and parkin recruitment, resulting in GAS replication in endothelial cells. IMPORTANCE In epithelial cells, GAS can be efficiently killed within the lysosome-fused autophaosome compartment. However, we previously showed that, in spite of LC-3 recruitment, the autophagic machinery is not sufficient for GAS killing in endothelial cells. In this report, we provide the first evidence that Gal-3, highly expressed in endothelial cells, blocks the tagging of ubiquitin to GAS by inhibiting recruitment of Gal-8 and parkin, leading to an enhancement of GAS replication. We also provide the first demonstration that Gal-8 can interact with parkin, the critical E3 ligase, for resistance to intracellular bacteria by facilitating the decoration of bacteria with ubiquitin chains. Our findings reveal that differential levels of Gal-3 and Gal-8 expression and recruitment to GAS between epithelial cells and endothelial cells may contribute to the different outcomes of GAS elimination or survival and growth of GAS in these two types of cells. Copyright © 2017 Cheng et al.

The tyrosine kinase Stitcher activates Grainy head and epidermal wound healing in Drosophila.

PubMed

Wang, Shenqiu; Tsarouhas, Vasilios; Xylourgidis, Nikos; Sabri, Nafiseh; Tiklová, Katarína; Nautiyal, Naumi; Gallio, Marco; Samakovlis, Christos

2009-07-01

Epidermal injury initiates a cascade of inflammation, epithelial remodelling and integument repair at wound sites. The regeneration of the extracellular barrier and damaged tissue repair rely on the precise orchestration of epithelial responses triggered by the injury. Grainy head (Grh) transcription factors induce gene expression to crosslink the extracellular barrier in wounded flies and mice. However, the activation mechanisms and functions of Grh factors in re-epithelialization remain unknown. Here we identify stitcher (stit), a new Grh target in Drosophila melanogaster. stit encodes a Ret-family receptor tyrosine kinase required for efficient epidermal wound healing. Live imaging analysis reveals that Stit promotes actin cable assembly during wound re-epithelialization. Stit activation also induces extracellular signal-regulated kinase (ERK) phosphorylation along with the Grh-dependent expression of stit and barrier repair genes at the wound sites. The transcriptional stimulation of stit on injury triggers a positive feedback loop increasing the magnitude of epithelial responses. Thus, Stit activation upon wounding coordinates cytoskeletal rearrangements and the level of Grh-mediated transcriptional wound responses.

ZBP1/DAI is an innate sensor of influenza virus triggering the NLRP3 inflammasome and programmed cell death pathways

PubMed Central

Kuriakose, Teneema; Man, Si Ming; Malireddi, R.K. Subbarao; Karki, Rajendra; Kesavardhana, Sannula; Place, David E.; Neale, Geoffrey; Vogel, Peter; Kanneganti, Thirumala-Devi

2016-01-01

The interferon-inducible protein Z-DNA binding protein 1 (ZBP1, also known as DNA-dependent activator of IFN-regulatory factors (DAI) and DLM-1) was identified as a dsDNA sensor, which instigates innate immune responses. However, this classification has been disputed and whether ZBP1 functions as a pathogen sensor during an infection has remained unknown. Herein, we demonstrated ZBP1-mediated sensing of the influenza A virus (IAV) proteins NP and PB1, triggering cell death and inflammatory responses via the RIPK1–RIPK3–Caspase-8 axis. ZBP1 regulates NLRP3 inflammasome activation as well as induction of apoptosis, necroptosis and pyroptosis in IAV-infected cells. Importantly, ZBP1 deficiency protected mice from mortality during IAV infection owing to reduced inflammatory responses and epithelial damage. Overall, these findings indicate that ZBP1 is an innate immune sensor of IAV and highlight its importance in the pathogenesis of IAV infection. PMID:27917412

Chronic epithelial kidney injury molecule-1 expression causes murine kidney fibrosis.

PubMed

Humphreys, Benjamin D; Xu, Fengfeng; Sabbisetti, Venkata; Grgic, Ivica; Movahedi Naini, Said; Wang, Ningning; Chen, Guochun; Xiao, Sheng; Patel, Dhruti; Henderson, Joel M; Ichimura, Takaharu; Mou, Shan; Soeung, Savuth; McMahon, Andrew P; Kuchroo, Vijay K; Bonventre, Joseph V

2013-09-01

Acute kidney injury predisposes patients to the development of both chronic kidney disease and end-stage renal failure, but the molecular details underlying this important clinical association remain obscure. We report that kidney injury molecule-1 (KIM-1), an epithelial phosphatidylserine receptor expressed transiently after acute injury and chronically in fibrotic renal disease, promotes kidney fibrosis. Conditional expression of KIM-1 in renal epithelial cells (Kim1(RECtg)) in the absence of an injury stimulus resulted in focal epithelial vacuolization at birth, but otherwise normal tubule histology and kidney function. By 4 weeks of age, Kim1(RECtg) mice developed spontaneous and progressive interstitial kidney inflammation with fibrosis, leading to renal failure with anemia, proteinuria, hyperphosphatemia, hypertension, cardiac hypertrophy, and death, analogous to progressive kidney disease in humans. Kim1(RECtg) kidneys had elevated expression of proinflammatory monocyte chemotactic protein-1 (MCP-1) at early time points. Heterologous expression of KIM-1 in an immortalized proximal tubule cell line triggered MCP-1 secretion and increased MCP-1-dependent macrophage chemotaxis. In mice expressing a mutant, truncated KIM-1 polypeptide, experimental kidney fibrosis was ameliorated with reduced levels of MCP-1, consistent with a profibrotic role for native KIM-1. Thus, sustained KIM-1 expression promotes kidney fibrosis and provides a link between acute and recurrent injury with progressive chronic kidney disease.

Diesel exhaust particles (DEP) induce an early redox imbalance followed by an IL-6 mediated inflammatory response on human conjunctival epithelial cells.

PubMed

Lasagni Vitar, Romina M; Tau, Julia; Janezic, Natasha S; Tesone, Agustina I; Hvozda Arana, Ailen G; Reides, Claudia G; Berra, Alejandro; Ferreira, Sandra M; Llesuy, Susana F

2018-06-01

The aim of this study was to evaluate the time course of oxidative stress markers and inflammatory mediators in human conjunctival epithelial cells (IOBA-NHC) exposed to diesel exhaust particles (DEP) for 1, 3, and 24 h. Reactive oxygen species (ROS) production, lipid and protein oxidation, Nrf2 pathway activation, enzymatic antioxidants, glutathione (GSH) levels and synthesis, as well as cytokine release and cell proliferation were analyzed. Cells exposed to DEP showed an increase in ROS at all time points. The induction of NADPH oxidase-4 appeared later than mitochondrial superoxide anion production, when the cell also underwent a proinflammatory response mediated by IL-6. DEP exposure triggered the activation of Nrf2 in IOBA-NHC, as a strategy for increasing cellular antioxidant capacity. Antioxidant enzyme activities were significantly increased at early stages except for glutathione reductase (GR) that showed a significant decrease after a 3-h-incubation. GSH levels were found increased after 1 and 3 h of incubation with DEP, despite the increase in its consumption by the antioxidant enzymes as it works as a cofactor. GSH recycling and the de novo synthesis were responsible for the maintenance of its content at these time points, respectively. After 24 h, the decrease in GR and glutamate cysteine ligase as wells as the enhanced activity of glutathione peroxidase and glutathione S-transferase produced a depletion in the GSH pool. Lipid-peroxidation was found increased in cells exposed to DEP after 1-h-incubation, whereas protein oxidation was found increased in cells exposed to DEP after a 3-h-incubation that persisted after a longer exposure. Furthermore, DEP lead IOBA-NHC cells to hyperplasia after 1 and 3 h of incubation, but a decrease in cell proliferation was found after longer exposure. ROS production seems to be an earlier event triggered by DEP on IOBA-NHC, comparing to the proinflammatory response mediated by IL-6. Despite the fact that under short periods of exposure to DEP lipids and then proteins are targets of oxidative damage, the viability of the cells is not affected at early stages, since cell hyperplasia was detected as compensatory mechanism. Although after 24 h Nrf2 pathway is still enhanced, the epithelial cell capacity to maintain redox balance is exceeded. The antioxidant enzymes activation and the depleted GSH pool are not capable of counteracting the increased ROS production, leading to oxidative damage. Copyright © 2018 Elsevier Ltd. All rights reserved.

Mechanisms of lung endothelial barrier disruption induced by cigarette smoke: role of oxidative stress and ceramides.

PubMed

Schweitzer, Kelly S; Hatoum, Hadi; Brown, Mary Beth; Gupta, Mehak; Justice, Matthew J; Beteck, Besem; Van Demark, Mary; Gu, Yuan; Presson, Robert G; Hubbard, Walter C; Petrache, Irina

2011-12-01

The epithelial and endothelial cells lining the alveolus form a barrier essential for the preservation of the lung respiratory function, which is, however, vulnerable to excessive oxidative, inflammatory, and apoptotic insults. Whereas profound breaches in this barrier function cause pulmonary edema, more subtle changes may contribute to inflammation. The mechanisms by which cigarette smoke (CS) exposure induce lung inflammation are not fully understood, but an early alteration in the epithelial barrier function has been documented. We sought to investigate the occurrence and mechanisms by which soluble components of mainstream CS disrupt the lung endothelial cell barrier function. Using cultured primary rat microvascular cell monolayers, we report that CS induces endothelial cell barrier disruption in a dose- and time-dependent manner of similar magnitude to that of the epithelial cell barrier. CS exposure triggered a mechanism of neutral sphingomyelinase-mediated ceramide upregulation and p38 MAPK and JNK activation that were oxidative stress dependent and that, along with Rho kinase activation, mediated the endothelial barrier dysfunction. The morphological changes in endothelial cell monolayers induced by CS included actin cytoskeletal rearrangement, junctional protein zonula occludens-1 loss, and intercellular gap formation, which were abolished by the glutathione modulator N-acetylcysteine and ameliorated by neutral sphingomyelinase inhibition. The direct application of ceramide recapitulated the effects of CS, by disrupting both endothelial and epithelial cells barrier, by a mechanism that was redox and apoptosis independent and required Rho kinase activation. Furthermore, ceramide induced dose-dependent alterations of alveolar microcirculatory barrier in vivo, measured by two-photon excitation microscopy in the intact rat. In conclusion, soluble components of CS have direct endothelial barrier-disruptive effects that could be ameliorated by glutathione modulators or by inhibitors of neutral sphingomyelinase, p38 MAPK, JNK, and Rho kinase. Amelioration of endothelial permeability may alleviate lung and systemic vascular dysfunction associated with smoking-related chronic obstructive lung diseases.

Structural centrosome aberrations sensitize polarized epithelia to basal cell extrusion.

PubMed

Ganier, Olivier; Schnerch, Dominik; Nigg, Erich A

2018-06-01

Centrosome aberrations disrupt tissue architecture and may confer invasive properties to cancer cells. Here we show that structural centrosome aberrations, induced by overexpression of either Ninein-like protein (NLP) or CEP131/AZI1, sensitize polarized mammalian epithelia to basal cell extrusion. While unperturbed epithelia typically dispose of damaged cells through apical dissemination into luminal cavities, certain oncogenic mutations cause a switch in directionality towards basal cell extrusion, raising the potential for metastatic cell dissemination. Here we report that NLP-induced centrosome aberrations trigger the preferential extrusion of damaged cells towards the basal surface of epithelial monolayers. This switch in directionality from apical to basal dissemination coincides with a profound reorganization of the microtubule cytoskeleton, which in turn prevents the contractile ring repositioning that is required to support extrusion towards the apical surface. While the basal extrusion of cells harbouring NLP-induced centrosome aberrations requires exogenously induced cell damage, structural centrosome aberrations induced by excess CEP131 trigger the spontaneous dissemination of dying cells towards the basal surface from MDCK cysts. Thus, similar to oncogenic mutations, structural centrosome aberrations can favour basal extrusion of damaged cells from polarized epithelia. Assuming that additional mutations may promote cell survival, this process could sensitize epithelia to disseminate potentially metastatic cells. © 2018 The Authors.

A subset of anti-rotavirus antibodies directed against the viral protein VP7 predicts the onset of celiac disease and induces typical features of the disease in the intestinal epithelial cell line T84.

PubMed

Dolcino, Marzia; Zanoni, Giovanna; Bason, Caterina; Tinazzi, Elisa; Boccola, Elisa; Valletta, Enrico; Contreas, Giovanna; Lunardi, Claudio; Puccetti, Antonio

2013-07-01

Celiac disease (CD) is an autoimmune disorder of the small intestine triggered by environmental factors in genetically predisposed individuals. A strong association between type 1 diabetes (T1DM) and CD has been reported. We have previously shown that rotavirus infection may be involved in the pathogenesis of CD through a mechanism of molecular mimicry. Indeed, we identified a subset of anti-transglutaminase IgA antibodies that recognize the rotavirus viral protein VP7. In this study, we aimed at evaluating whether such antibodies may predict the onset of CD in children affected by T1DM. Moreover, to further analyze the link between rotavirus infection and pathogenesis of CD, we analyzed the effect of anti-rotavirus VP7 antibodies on T84 intestinal epithelial cells using the gene-array technique, complemented by the analysis of molecules secreted in the supernatant of stimulated cells. We found that anti-rotavirus VP7 antibodies are present in the vast majority (81%) of T1DM-CD tested sera, but are detectable also in a fraction (27%) of T1DM children without CD. Moreover, we found that anti-rotavirus VP7 antibodies are present before the CD onset, preceding the detection of anti-tTG and anti-endomysium antibodies. The gene-array analysis showed that purified anti-rotavirus VP7 antibodies modulate genes that are involved in apoptosis, inflammation, and alteration of the epithelial barrier integrity in intestinal epithelial cells, all typical features of CD. Taken together, these new data further support the involvement of rotavirus infection in the pathogenesis of CD and suggest a predictive role of anti-rotavirus VP7 antibodies.

Comprehensive evaluation of poly(I:C) induced inflammatory response in an airway epithelial model

PubMed Central

Lever, Amanda R; Park, Hyoungshin; Mulhern, Thomas J; Jackson, George R; Comolli, James C; Borenstein, Jeffrey T; Hayden, Patrick J; Prantil-Baun, Rachelle

2015-01-01

Respiratory viruses invade the upper airway of the lung, triggering a potent immune response that often exacerbates preexisting conditions such as asthma and COPD. Poly(I:C) is a synthetic analog of viral dsRNA that induces the characteristic inflammatory response associated with viral infection, such as loss of epithelial integrity, and increased production of mucus and inflammatory cytokines. Here, we explore the mechanistic responses to poly(I:C) in a well-defined primary normal human bronchial epithelial (NHBE) model that recapitulates in vivo functions and responses. We developed functional and quantifiable methods to evaluate the physiology of our model in both healthy and inflamed states. Through gene and protein expression, we validated the differentiation state and population of essential cell subtypes (i.e., ciliated, goblet, club, and basal cells) as compared to the human lung. Assays for total mucus production, cytokine secretion, and barrier function were used to evaluate in vitro physiology and response to viral insult. Cells were treated apically with poly(I:C) and evaluated 48 h after induction. Results revealed a dose-dependent increase in goblet cell differentiation, as well as, an increase in mucus production relative to controls. There was also a dose-dependent increase in secretion of IL-6, IL-8, TNF-α, and RANTES. Epithelial barrier function, as measured by TEER, was maintained at 1501 ± 355 Ω*cm² postdifferentiation, but dropped significantly when challenged with poly(I:C). This study provides first steps toward a well-characterized model with defined functional methods for understanding dsRNA stimulated inflammatory responses in a physiologically relevant manner. PMID:25847914

Cystine addiction of triple-negative breast cancer associated with EMT augmented death signaling.

PubMed

Tang, X; Ding, C-K; Wu, J; Sjol, J; Wardell, S; Spasojevic, I; George, D; McDonnell, D P; Hsu, D S; Chang, J T; Chi, J-T

2017-07-27

Despite the advances in the diagnosis and treatment of breast cancer, breast cancers still cause significant mortality. For some patients, especially those with triple-negative breast cancer, current treatments continue to be limited and ineffective. Therefore, there remains an unmet need for a novel therapeutic approach. One potential strategy is to target the altered metabolic state that is rewired by oncogenic transformation. Specifically, this rewiring may render certain outside nutrients indispensable. To identify such a nutrient, we performed a nutrigenetic screen by removing individual amino acids to identify possible addictions across a panel of breast cancer cells. This screen revealed that cystine deprivation triggered rapid programmed necrosis, but not apoptosis, in the basal-type breast cancer cells mostly seen in TNBC tumors. In contrast, luminal-type breast cancer cells are cystine-independent and exhibit little death during cystine deprivation. The cystine addiction phenotype is associated with a higher level of cystine-deprivation signatures noted in the basal type breast cancer cells and tumors. We found that the cystine-addicted breast cancer cells and tumors have strong activation of TNFα and MEKK4-p38-Noxa pathways that render them susceptible to cystine deprivation-induced necrosis. Consistent with this model, silencing of TNFα and MEKK4 dramatically reduces cystine-deprived death. In addition, the cystine addiction phenotype can be abrogated in the cystine-addictive cells by miR-200c, which converts the mesenchymal-like cells to adopt epithelial features. Conversely, the introduction of inducers of epithelial-mesenchymal transition (EMT) in cystine-independent breast cancer cells conferred the cystine-addiction phenotype by modulating the signaling components of cystine addiction. Together, our data reveal that cystine-addiction is associated with EMT in breast cancer during tumor progression. These findings provide the genetic and mechanistic basis to explain how cystine deprivation triggers necrosis by activating pre-existing oncogenic pathways in cystine-addicted TNBC with prominent mesenchymal features.

The Role of Interleukin-1 and Interleukin-18 in Pro-Inflammatory and Anti-Viral Responses to Rhinovirus in Primary Bronchial Epithelial Cells

PubMed Central

Kay, Linda; Parker, Lisa C.; Sabroe, Ian; Sleeman, Matthew A.; Briend, Emmanuel; Finch, Donna K.

2013-01-01

Human Rhinovirus (HRV) is associated with acute exacerbations of chronic respiratory disease. In healthy individuals, innate viral recognition pathways trigger release of molecules with direct anti-viral activities and pro-inflammatory mediators which recruit immune cells to support viral clearance. Interleukin-1alpha (IL-1α), interleukin-1beta (IL-1β) and interleukin-18 (IL-18) have critical roles in the establishment of neutrophilic inflammation, which is commonly seen in airways viral infection and thought to be detrimental in respiratory disease. We therefore investigated the roles of these molecules in HRV infection of primary human epithelial cells. We found that all three cytokines were released from infected epithelia. Release of these cytokines was not dependent on cell death, and only IL-1β and IL-18 release was dependent on caspase-1 catalytic activity. Blockade of IL-1 but not IL-18 signaling inhibited up-regulation of pro-inflammatory mediators and neutrophil chemoattractants but had no effect on virus induced production of interferons and interferon-inducible genes, measured at both mRNA and protein level. Similar level of virus mRNA was detected with and without IL-1RI blockade. Hence IL-1 signaling, potentially involving both IL-1β and IL-1α, downstream of viral recognition plays a key role in induction of pro-inflammatory signals and potentially in recruitment and activation of immune cells in response to viral infection instigated by the epithelial cells, whilst not participating in direct anti-viral responses. PMID:23723976

Human rhinovirus-induced ISG15 selectively modulates epithelial antiviral immunity

PubMed Central

Zaheer, R S; Wiehler, S; Hudy, M H; Traves, S L; Pelikan, J B; Leigh, R; Proud, D

2014-01-01

Human rhinovirus (HRV) infections trigger exacerbations of lower airway diseases. HRV infects human airway epithelial cells and induces proinflammatory and antiviral molecules that regulate the response to HRV infection. Interferon (IFN)-stimulated gene of 15 kDa (ISG15) has been shown to regulate other viruses. We now show that HRV-16 infection induces both intracellular epithelial ISG15 expression and ISG15 secretion in vitro. Moreover, ISG15 protein levels increased in nasal secretions of subjects with symptomatic HRV infections. HRV-16-induced ISG15 expression is transcriptionally regulated via an IFN regulatory factor pathway. ISG15 does not directly alter HRV replication but does modulate immune signaling via the viral sensor protein RIG-I to impact production of CXCL10, which has been linked to innate immunity to viruses. Extracellular ISG15 also alters CXCL10 production. We conclude that ISG15 has a complex role in host defense against HRV infection, and that additional studies are needed to clarify the role of this molecule. PMID:24448099

Light induced cytosolic drug delivery from liposomes with gold nanoparticles.

PubMed

Lajunen, Tatu; Viitala, Lauri; Kontturi, Leena-Stiina; Laaksonen, Timo; Liang, Huamin; Vuorimaa-Laukkanen, Elina; Viitala, Tapani; Le Guével, Xavier; Yliperttula, Marjo; Murtomäki, Lasse; Urtti, Arto

2015-04-10

Externally triggered drug release at defined targets allows site- and time-controlled drug treatment regimens. We have developed liposomal drug carriers with encapsulated gold nanoparticles for triggered drug release. Light energy is converted to heat in the gold nanoparticles and released to the lipid bilayers. Localized temperature increase renders liposomal bilayers to be leaky and triggers drug release. The aim of this study was to develop a drug releasing system capable of releasing its cargo to cell cytosol upon triggering with visible and near infrared light signals. The liposomes were formulated using either heat-sensitive or heat- and pH-sensitive lipid compositions with star or rod shaped gold nanoparticles. Encapsulated fluorescent probe, calcein, was released from the liposomes after exposure to the light. In addition, the pH-sensitive formulations showed a faster drug release in acidic conditions than in neutral conditions. The liposomes were internalized into human retinal pigment epithelial cells (ARPE-19) and human umbilical vein endothelial cells (HUVECs) and did not show any cellular toxicity. The light induced cytosolic delivery of calcein from the gold nanoparticle containing liposomes was shown, whereas no cytosolic release was seen without light induction or without gold nanoparticles in the liposomes. The light activated liposome formulations showed a controlled content release to the cellular cytosol at a specific location and time. Triggering with visual and near infrared light allows good tissue penetration and safety, and the pH-sensitive liposomes may enable selective drug release in the intracellular acidic compartments (endosomes, lysosomes). Thus, light activated liposomes with gold nanoparticles are an attractive option for time- and site-specific drug delivery into the target cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Epithelial architectural destruction is necessary for bone marrow derived cell contribution to regenerating prostate epithelium.

PubMed

Palapattu, Ganesh S; Meeker, Alan; Harris, Timothy; Collector, Michael I; Sharkis, Saul J; DeMarzo, Angelo M; Warlick, Christopher; Drake, Charles G; Nelson, William G

2006-08-01

Using various nonphysiological tissue injury/repair models numerous studies have demonstrated the capacity of bone marrow derived cells to contribute to the repopulation of epithelial tissues following damage. To investigate whether this phenomenon might also occur during periods of physiological tissue degeneration/regeneration we compared the ability of bone marrow derived cells to rejuvenate the prostate gland in mice that were castrated and then later treated with dihydrotestosterone vs mice with prostate epithelium that had been damaged by lytic virus infection. Using allogenic bone marrow grafts from female donor transgenic mice expressing green fluorescent protein transplanted into lethally irradiated males we were able to assess the contributions of bone marrow derived cells to recovery of the prostatic epithelium in 2 distinct systems, including 1) surgical castration followed 1 week later by dihydrotestosterone replacement and 2) intraprostatic viral injection. Eight to 10-week-old male C57/Bl6 mice were distributed among bone marrow donor-->recipient/prostate injury groups, including 5 with C57/Bl6-->C57/Bl6/no injury, 3 with green fluorescent protein-->C57/Bl6/no injury, 3 with green fluorescent protein-->C57/Bl6/vehicle injection, 4 with green fluorescent protein-->C57/Bl6/virus injection and 3 each with green fluorescent protein-->C57/Bl6/castration without and with dihydrotestosterone, respectively. Prostate tissues were harvested 3 weeks after dihydrotestosterone replacement or 14 days following intraprostatic viral injection. Prostate tissue immunofluorescence was performed with antibodies against the epithelial marker cytokeratin 5/8, the hematopoietic marker CD45 and green fluorescent protein. Mice that sustained prostate injury from vaccinia virus infection with concomitant severe inflammation and glandular disruption showed evidence of bone marrow derived cell reconstitution of prostate epithelium, that is approximately 4% of all green fluorescent protein positive cells in the epithelial compartment 14 days after injury expressed cytokeratin 5/8, similar to the proportion of green fluorescent protein positive cells in the prostate that no longer expressed the hematopoietic marker CD45. When prostatic degeneration/regeneration was triggered by androgen deprivation and reintroduction, no green fluorescent protein positive prostate epithelial cells were detected. These findings are consistent with a requirement for inflammation associated architectural destruction for the bone marrow derived cell contribution to the regeneration of prostate epithelium.

BRD4 mediates NF-κB-dependent epithelial-mesenchymal transition and pulmonary fibrosis via transcriptional elongation

PubMed Central

Zhao, Yingxin; Sun, Hong; Zhang, Yueqing; Yang, Jun; Brasier, Allan R.

2016-01-01

Chronic epithelial injury triggers a TGF-β-mediated cellular transition from normal epithelium into a mesenchymal-like state that produces subepithelial fibrosis and airway remodeling. Here we examined how TGF-β induces the mesenchymal cell state and determined its mechanism. We observed that TGF-β stimulation activates an inflammatory gene program controlled by the NF-κB/RelA signaling pathway. In the mesenchymal state, NF-κB-dependent immediate-early genes accumulate euchromatin marks and processive RNA polymerase. This program of immediate-early genes is activated by enhanced expression, nuclear translocation, and activating phosphorylation of the NF-κB/RelA transcription factor on Ser276, mediated by a paracrine signal. Phospho-Ser276 RelA binds to the BRD4/CDK9 transcriptional elongation complex, activating the paused RNA Pol II by phosphorylation on Ser2 in its carboxy-terminal domain. RelA-initiated transcriptional elongation is required for expression of the core epithelial-mesenchymal transition transcriptional regulators SNAI1, TWIST1, and ZEB1 and mesenchymal genes. Finally, we observed that pharmacological inhibition of BRD4 can attenuate experimental lung fibrosis induced by repetitive TGF-β challenge in a mouse model. These data provide a detailed mechanism for how activated NF-κB and BRD4 control epithelial-mesenchymal transition initiation and transcriptional elongation in model airway epithelial cells in vitro and in a murine pulmonary fibrosis model in vivo. Our data validate BRD4 as an in vivo target for the treatment of pulmonary fibrosis associated with inflammation-coupled remodeling in chronic lung diseases. PMID:27793799

High Degree of Overlap between Responses to a Virus and to the House Dust Mite Allergen in Airway Epithelial Cells

PubMed Central

Golebski, Korneliusz; Luiten, Silvia; van Egmond, Danielle; de Groot, Esther; Röschmann, Kristina Irene Lisolette; Fokkens, Wytske Johanna; van Drunen, Cornelis Maria

2014-01-01

Background Airway epithelium is widely considered to play an active role in immune responses through its ability to detect changes in the environment and to generate a microenvironment for immune competent cells. Therefore, besides its role as a physical barrier, epithelium affects the outcome of the immune response by the production of various pro-inflammatory mediators. Methods We stimulated airway epithelial cells with viral double stranded RNA analogue poly(I:C) or with house dust mite in a time course of 24 hours. In order to determine cytokines production by stimulated cells, we performed multiplex enzyme linked immunosorbant assay (ELISA). Results We demonstrate that the temporal pattern of the genes that respond to virus exposure in airway epithelium resembles to a significant degree their pattern of response to HDM. The gene expression pattern of EGR1, DUSP1, FOSL1, JUN, MYC, and IL6 is rather similar after viral (poly(I:C)) and HDM exposure. However, both triggers also induce a specific response (e.g. ATF3, FOS, and NFKB1). We confirmed these data by showing that epithelial cells produce a variety of similar mediators in response to both poly(I:C) and HDM challenge (IL1-RA, IL-17, IFN-α and MIP1-α), sometimes with a quantitative difference in response (IL2-R, IL-6, IL-8, MCP-1, MIG, and HGF). Interestingly, only four mediators (IL-12, IP-10, RANTES and VEGF) where up-regulated specifically by poly(I:C) and not by HDM. Additionally, we report that pre-exposure to HDM deregulates production of cytokines and mediators in response to poly(I:C). Conclusions Epithelial cells responses to the HDM-allergen and a virus strongly resemble both in gene expression and in protein level explaining why these two responses may affect each other. PMID:24498371

High degree of overlap between responses to a virus and to the house dust mite allergen in airway epithelial cells.

PubMed

Golebski, Korneliusz; Luiten, Silvia; van Egmond, Danielle; de Groot, Esther; Röschmann, Kristina Irene Lisolette; Fokkens, Wytske Johanna; van Drunen, Cornelis Maria

2014-01-01

Airway epithelium is widely considered to play an active role in immune responses through its ability to detect changes in the environment and to generate a microenvironment for immune competent cells. Therefore, besides its role as a physical barrier, epithelium affects the outcome of the immune response by the production of various pro-inflammatory mediators. We stimulated airway epithelial cells with viral double stranded RNA analogue poly(I:C) or with house dust mite in a time course of 24 hours. In order to determine cytokines production by stimulated cells, we performed multiplex enzyme linked immunosorbant assay (ELISA). We demonstrate that the temporal pattern of the genes that respond to virus exposure in airway epithelium resembles to a significant degree their pattern of response to HDM. The gene expression pattern of EGR1, DUSP1, FOSL1, JUN, MYC, and IL6 is rather similar after viral (poly(I:C)) and HDM exposure. However, both triggers also induce a specific response (e.g. ATF3, FOS, and NFKB1). We confirmed these data by showing that epithelial cells produce a variety of similar mediators in response to both poly(I:C) and HDM challenge (IL1-RA, IL-17, IFN-α and MIP1-α), sometimes with a quantitative difference in response (IL2-R, IL-6, IL-8, MCP-1, MIG, and HGF). Interestingly, only four mediators (IL-12, IP-10, RANTES and VEGF) where up-regulated specifically by poly(I:C) and not by HDM. Additionally, we report that pre-exposure to HDM deregulates production of cytokines and mediators in response to poly(I:C). Epithelial cells responses to the HDM-allergen and a virus strongly resemble both in gene expression and in protein level explaining why these two responses may affect each other.

Compound A, a Selective Glucocorticoid Receptor Modulator, Enhances Heat Shock Protein Hsp70 Gene Promoter Activation

PubMed Central

Beck, Ilse M.; Drebert, Zuzanna J.; Hoya-Arias, Ruben; Bahar, Ali A.; Devos, Michael; Clarisse, Dorien; Desmet, Sofie; Bougarne, Nadia; Ruttens, Bart; Gossye, Valerie; Denecker, Geertrui; Lievens, Sam; Bracke, Marc; Tavernier, Jan; Declercq, Wim; Gevaert, Kris; Berghe, Wim Vanden; Haegeman, Guy; De Bosscher, Karolien

2013-01-01

Compound A possesses glucocorticoid receptor (GR)-dependent anti-inflammatory properties. Just like classical GR ligands, Compound A can repress NF-κB-mediated gene expression. However, the monomeric Compound A-activated GR is unable to trigger glucocorticoid response element-regulated gene expression. The heat shock response potently activates heat shock factor 1 (HSF1), upregulates Hsp70, a known GR chaperone, and also modulates various aspects of inflammation. We found that the selective GR modulator Compound A and heat shock trigger similar cellular effects in A549 lung epithelial cells. With regard to their anti-inflammatory mechanism, heat shock and Compound A are both able to reduce TNF-stimulated IκBα degradation and NF-κB p65 nuclear translocation. We established an interaction between Compound A-activated GR and Hsp70, but remarkably, although the presence of the Hsp70 chaperone as such appears pivotal for the Compound A-mediated inflammatory gene repression, subsequent novel Hsp70 protein synthesis is uncoupled from an observed CpdA-induced Hsp70 mRNA upregulation and hence obsolete in mediating CpdA’s anti-inflammatory effect. The lack of a Compound A-induced increase in Hsp70 protein levels in A549 cells is not mediated by a rapid proteasomal degradation of Hsp70 or by a Compound A-induced general block on translation. Similar to heat shock, Compound A can upregulate transcription of Hsp70 genes in various cell lines and BALB/c mice. Interestingly, whereas Compound A-dependent Hsp70 promoter activation is GR-dependent but HSF1-independent, heat shock-induced Hsp70 expression alternatively occurs in a GR-independent and HSF1-dependent manner in A549 lung epithelial cells. PMID:23935933

Diverse Targets of β-Catenin during the Epithelial-Mesenchymal Transition Define Cancer Stem Cells and Predict Disease Relapse.

PubMed

Chang, Yi-Wen; Su, Ying-Jhen; Hsiao, Michael; Wei, Kuo-Chen; Lin, Wei-Hsin; Liang, Chi-Lung; Chen, Shin-Cheh; Lee, Jia-Lin

2015-08-15

Wnt signaling contributes to the reprogramming and maintenance of cancer stem cell (CSC) states that are activated by epithelial-mesenchymal transition (EMT). However, the mechanistic relationship between EMT and the Wnt pathway in CSC is not entirely clear. Chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) indicated that EMT induces a switch from the β-catenin/E-cadherin/Sox15 complex to the β-catenin/Twist1/TCF4 complex, the latter of which then binds to CSC-related gene promoters. Tandem coimmunoprecipitation and re-ChIP experiments with epithelial-type cells further revealed that Sox15 associates with the β-catenin/E-cadherin complex, which then binds to the proximal promoter region of CASP3. Through this mechanism, Twist1 cleavage is triggered to regulate a β-catenin-elicited promotion of the CSC phenotype. During EMT, we documented that Twist1 binding to β-catenin enhanced the transcriptional activity of the β-catenin/TCF4 complex, including by binding to the proximal promoter region of ABCG2, a CSC marker. In terms of clinical application, our definition of a five-gene CSC signature (nuclear β-catenin(High)/nuclear Twist1(High)/E-cadherin(Low)/Sox15(Low)/CD133(High)) may provide a useful prognostic marker for human lung cancer. ©2015 American Association for Cancer Research.

Photoaging and skin cancer: Is the inflammasome the missing link?

PubMed

Awad, Fawaz; Assrawi, Eman; Louvrier, Camille; Jumeau, Claire; Giurgea, Irina; Amselem, Serge; Karabina, Sonia-Athina

2018-03-12

Photoaging and epithelial skin tumorigenesis are complex processes triggered mainly by UV radiation from chronic sun exposure. This leads to DNA damage and reactive oxygen species (ROS) production, which initiate an inflammatory response that alters cell structure and function. Changes in cell homeostasis and ROS production activate intracellular multiprotein platforms called inflammasomes. Inflammasomes nucleate around cytoplasmic receptors mainly of the NLR (nucleotide-binding domain and leucine-rich repeat) family and regulate caspase-1-dependant secretion of pro-inflammatory interleukin (IL)1β and IL18 cytokines, and an inflammatory form of death named pyroptosis. NLRP1 inflammasomes have taken centre stage in skin biology, as mutations in NLRP1 underlie the genetic etiology of dermatological diseases and increase the susceptibility to skin cancer. Targeting inflammasome(s) might be an important approach to improve skin inflammation, photoaging and reduce the risk of epithelial skin tumorigenesis. In this context, we discuss the potential implication of NLRP1 and NLRP3 inflammasomes. Copyright © 2018 Elsevier B.V. All rights reserved.

Evidence of Epstein-Barr Virus Association with Gastric Cancer and Non-Atrophic Gastritis

PubMed Central

Martínez-López, Juan L.E.; Torres, Javier; Camorlinga-Ponce, Margarita; Mantilla, Alejandra; Leal, Yelda A.; Fuentes-Pananá, Ezequiel M.

2014-01-01

Different lines of evidence support an association between Epstein-Barr virus (EBV) and gastric cancer (GC). The main understood risk factor to develop GC is infection by Helicobacter pylori (H. pylori), which triggers a local inflammatory response critical for progression from gastritis to GC. The role of EBV in early inflammatory gastric lesions has been poorly studied. A recent study proposed a cutoff value of 2000 EBV particles to identify patients with increased chances of infection of the gastric epithelium, which may favor the inflammatory process. To better understand the role of EBV in cancer progression, we analyzed 75 samples of GC, 147 control samples of non-tumor gastric tissue derived from GC patients and 75 biopsies from patients with non-atrophic gastritis (NAG). A first-round PCR was used for EBV detection in tumor and non-tumor controls and a more sensitive nested PCR for gastritis samples; both PCRs had lower detection limits above the proposed cutoff value. With this strategy 10.67% of GC, 1.3% of non-tumor controls and 8% of gastritis samples were found positive. An EBER1 in situ hybridization showed EBV infection of epithelial cells in GC and in a third of NAG samples, while in the other NAGs infection was restricted to the mononuclear cell infiltrate. EBV-positive GCs were enriched in lace and cribriform patterns, while these rare patterns were not observed in EBV negative samples. Our results support a role for EBV in GC and early precursor lesions, either as directly oncogenic infecting epithelial cells or indirectly as an inflammatory trigger. PMID:24448220

Next-generation sequencing reveals low-dose effects of cationic dendrimers in primary human bronchial epithelial cells.

PubMed

Feliu, Neus; Kohonen, Pekka; Ji, Jie; Zhang, Yuning; Karlsson, Hanna L; Palmberg, Lena; Nyström, Andreas; Fadeel, Bengt

2015-01-27

Gene expression profiling has developed rapidly in recent years with the advent of deep sequencing technologies such as RNA sequencing (RNA Seq) and could be harnessed to predict and define mechanisms of toxicity of chemicals and nanomaterials. However, the full potential of these technologies in (nano)toxicology is yet to be realized. Here, we show that systems biology approaches can uncover mechanisms underlying cellular responses to nanomaterials. Using RNA Seq and computational approaches, we found that cationic poly(amidoamine) dendrimers (PAMAM-NH2) are capable of triggering down-regulation of cell-cycle-related genes in primary human bronchial epithelial cells at doses that do not elicit acute cytotoxicity, as demonstrated using conventional cell viability assays, while gene transcription was not affected by neutral PAMAM-OH dendrimers. The PAMAMs were internalized in an active manner by lung cells and localized mainly in lysosomes; amine-terminated dendrimers were internalized more efficiently when compared to the hydroxyl-terminated dendrimers. Upstream regulator analysis implicated NF-κB as a putative transcriptional regulator, and subsequent cell-based assays confirmed that PAMAM-NH2 caused NF-κB-dependent cell cycle arrest. However, PAMAM-NH2 did not affect cell cycle progression in the human A549 adenocarcinoma cell line. These results demonstrate the feasibility of applying systems biology approaches to predict cellular responses to nanomaterials and highlight the importance of using relevant (primary) cell models.

Circadian clock component REV-ERBα controls homeostatic regulation of pulmonary inflammation.

PubMed

Pariollaud, Marie; Gibbs, Julie E; Hopwood, Thomas W; Brown, Sheila; Begley, Nicola; Vonslow, Ryan; Poolman, Toryn; Guo, Baoqiang; Saer, Ben; Jones, D Heulyn; Tellam, James P; Bresciani, Stefano; Tomkinson, Nicholas Co; Wojno-Picon, Justyna; Cooper, Anthony Wj; Daniels, Dion A; Trump, Ryan P; Grant, Daniel; Zuercher, William; Willson, Timothy M; MacDonald, Andrew S; Bolognese, Brian; Podolin, Patricia L; Sanchez, Yolanda; Loudon, Andrew Si; Ray, David W

2018-06-01

Recent studies reveal that airway epithelial cells are critical pulmonary circadian pacemaker cells, mediating rhythmic inflammatory responses. Using mouse models, we now identify the rhythmic circadian repressor REV-ERBα as essential to the mechanism coupling the pulmonary clock to innate immunity, involving both myeloid and bronchial epithelial cells in temporal gating and determining amplitude of response to inhaled endotoxin. Dual mutation of REV-ERBα and its paralog REV-ERBβ in bronchial epithelia further augmented inflammatory responses and chemokine activation, but also initiated a basal inflammatory state, revealing a critical homeostatic role for REV-ERB proteins in the suppression of the endogenous proinflammatory mechanism in unchallenged cells. However, REV-ERBα plays the dominant role, as deletion of REV-ERBβ alone had no impact on inflammatory responses. In turn, inflammatory challenges cause striking changes in stability and degradation of REV-ERBα protein, driven by SUMOylation and ubiquitination. We developed a novel selective oxazole-based inverse agonist of REV-ERB, which protects REV-ERBα protein from degradation, and used this to reveal how proinflammatory cytokines trigger rapid degradation of REV-ERBα in the elaboration of an inflammatory response. Thus, dynamic changes in stability of REV-ERBα protein couple the core clock to innate immunity.

Group A Streptococcus tissue invasion by CD44-mediated cell signalling

NASA Astrophysics Data System (ADS)

Cywes, Colette; Wessels, Michael R.

2001-12-01

Streptococcus pyogenes (also known as group A Streptococcus, GAS), the agent of streptococcal sore throat and invasive soft-tissue infections, attaches to human pharyngeal or skin epithelial cells through specific recognition of its hyaluronic acid capsular polysaccharide by the hyaluronic-acid-binding protein CD44 (refs 1, 2). Because ligation of CD44 by hyaluronic acid can induce epithelial cell movement on extracellular matrix, we investigated whether molecular mimicry by the GAS hyaluronic acid capsule might induce similar cellular responses. Here we show that CD44-dependent GAS binding to polarized monolayers of human keratinocytes induced marked cytoskeletal rearrangements manifested by membrane ruffling and disruption of intercellular junctions. Transduction of the signal induced by GAS binding to CD44 on the keratinocyte surface involved Rac1 and the cytoskeleton linker protein ezrin, as well as tyrosine phosphorylation of cellular proteins. Studies of bacterial translocation in two models of human skin indicated that cell signalling triggered by interaction of the GAS capsule with CD44 opened intercellular junctions and promoted tissue penetration by GAS through a paracellular route. These results support a model of host cytoskeleton manipulation and tissue invasion by an extracellular bacterial pathogen.

Modification of Intestinal Microbiota and Its Consequences for Innate Immune Response in the Pathogenesis of Campylobacteriosis

PubMed Central

Heimesaat, Markus M.; Bereswill, Stefan; Tareen, Abdul Malik; Lugert, Raimond; Groß, Uwe; Zautner, Andreas E.

2013-01-01

Campylobacter jejuni is the leading cause of bacterial food-borne gastroenteritis in the world, and thus one of the most important public health concerns. The initial stage in its pathogenesis after ingestion is to overcome colonization resistance that is maintained by the human intestinal microbiota. But how it overcomes colonization resistance is unknown. Recently developed humanized gnotobiotic mouse models have provided deeper insights into this initial stage and host's immune response. These studies have found that a fat-rich diet modifies the composition of the conventional intestinal microbiota by increasing the Firmicutes and Proteobacteria loads while reducing the Actinobacteria and Bacteroidetes loads creating an imbalance that exposes the intestinal epithelial cells to adherence. Upon adherence, deoxycholic acid stimulates C. jejuni to synthesize Campylobacter invasion antigens, which invade the epithelial cells. In response, NF-κB triggers the maturation of dendritic cells. Chemokines produced by the activated dendritic cells initiate the clearance of C. jejuni cells by inducing the actions of neutrophils, B-lymphocytes, and various subsets of T-cells. This immune response causes inflammation. This review focuses on the progress that has been made on understanding the relationship between intestinal microbiota shift, establishment of C. jejuni infection, and consequent immune response. PMID:24324507

Apoptosis of Corneal Epithelial Cells Caused by Ultraviolet B-induced Loss of K+ is Inhibited by Ba2+

PubMed Central

Glupker, Courtney D.; Boersma, Peter M.; Schotanus, Mark P.; Haarsma, Loren D.; Ubels, John L.

2017-01-01

UVB exposure at ambient outdoor levels triggers rapid K+ loss and apoptosis in human corneal limbal epithelial (HCLE) cells cultured in medium containing 5.5 mM K+, but considerably less apoptosis occurs when the medium contains the high K+ concentration that is present in tears (25 mM). Since Ba2+ blocks several K+ channels, we tested whether Ba2+-sensitive K+ channels are responsible for some or all of the UVB-activated K+ loss and subsequent activation of the caspase cascade and apoptosis. Corneal epithelial cells in culture were exposed to UVB at 80 or 150 mJ/cm2. Patch-clamp recording was used to measure UVB-induced K+ currents. Caspase-activity and TUNEL assays were performed on HCLE cells exposed to UVB followed by incubation in the presence or absence of Ba2+. K+ currents were activated in HCLE cells following UVB-exposure. These currents were reversibly blocked by 5 mM Ba2+. When HCLE cells were incubated with 5 mM Ba2+ after exposure to UVB, activation of caspases-9, -8, and -3 and DNA fragmentation were significantly decreased. The data confirm that UVB-induced K+ current activation and loss of intracellular K+ leads to activation of the caspase cascade and apoptosis. Extracellular Ba2+ inhibits UVB-induced apoptosis by preventing loss of intracellular K+ when K+ channels are activated. Ba2+ therefore has effects similar to elevated extracellular K+ in protecting HCLE cells from UVB-induced apoptosis. This supports our overall hypothesis that elevated K+ in tears contributes to protection of the corneal epithelium from adverse effects of ambient outdoor UVB. PMID:27189864

Human Keratinocytes That Express hTERT and Also Bypass a p16INK4a-Enforced Mechanism That Limits Life Span Become Immortal yet Retain Normal Growth and Differentiation Characteristics

PubMed Central

Dickson, Mark A.; Hahn, William C.; Ino, Yasushi; Ronfard, Vincent; Wu, Jenny Y.; Weinberg, Robert A.; Louis, David N.; Li, Frederick P.; Rheinwald, James G.

2000-01-01

Normal human cells exhibit a limited replicative life span in culture, eventually arresting growth by a process termed senescence. Progressive telomere shortening appears to trigger senescence in normal human fibroblasts and retinal pigment epithelial cells, as ectopic expression of the telomerase catalytic subunit, hTERT, immortalizes these cell types directly. Telomerase expression alone is insufficient to enable certain other cell types to evade senescence, however. Such cells, including keratinocytes and mammary epithelial cells, appear to require loss of the pRB/p16INK4a cell cycle control mechanism in addition to hTERT expression to achieve immortality. To investigate the relationships among telomerase activity, cell cycle control, senescence, and differentiation, we expressed hTERT in two epithelial cell types, keratinocytes and mesothelial cells, and determined the effect on proliferation potential and on the function of cell-type-specific growth control and differentiation systems. Ectopic hTERT expression immortalized normal mesothelial cells and a premalignant, p16INK4a-negative keratinocyte line. In contrast, when four keratinocyte strains cultured from normal tissue were transduced to express hTERT, they were incompletely rescued from senescence. After reaching the population doubling limit of their parent cell strains, hTERT+ keratinocytes entered a slow growth phase of indefinite length, from which rare, rapidly dividing immortal cells emerged. These immortal cell lines frequently had sustained deletions of the CDK2NA/INK4A locus or otherwise were deficient in p16INK4a expression. They nevertheless typically retained other keratinocyte growth controls and differentiated normally in culture and in xenografts. Thus, keratinocyte replicative potential is limited by a p16INK4a-dependent mechanism, the activation of which can occur independent of telomere length. Abrogation of this mechanism together with telomerase expression immortalizes keratinocytes without affecting other major growth control or differentiation systems. PMID:10648628

Core epithelial-to-mesenchymal transition interactome gene-expression signature is associated with claudin-low and metaplastic breast cancer subtypes.

PubMed

Taube, Joseph H; Herschkowitz, Jason I; Komurov, Kakajan; Zhou, Alicia Y; Gupta, Supriya; Yang, Jing; Hartwell, Kimberly; Onder, Tamer T; Gupta, Piyush B; Evans, Kurt W; Hollier, Brett G; Ram, Prahlad T; Lander, Eric S; Rosen, Jeffrey M; Weinberg, Robert A; Mani, Sendurai A

2010-08-31

The epithelial-to-mesenchymal transition (EMT) produces cancer cells that are invasive, migratory, and exhibit stem cell characteristics, hallmarks of cells that have the potential to generate metastases. Inducers of the EMT include several transcription factors (TFs), such as Goosecoid, Snail, and Twist, as well as the secreted TGF-beta1. Each of these factors is capable, on its own, of inducing an EMT in the human mammary epithelial (HMLE) cell line. However, the interactions between these regulators are poorly understood. Overexpression of each of the above EMT inducers up-regulates a subset of other EMT-inducing TFs, with Twist, Zeb1, Zeb2, TGF-beta1, and FOXC2 being commonly induced. Up-regulation of Slug and FOXC2 by either Snail or Twist does not depend on TGF-beta1 signaling. Gene expression signatures (GESs) derived by overexpressing EMT-inducing TFs reveal that the Twist GES and Snail GES are the most similar, although the Goosecoid GES is the least similar to the others. An EMT core signature was derived from the changes in gene expression shared by up-regulation of Gsc, Snail, Twist, and TGF-beta1 and by down-regulation of E-cadherin, loss of which can also trigger an EMT in certain cell types. The EMT core signature associates closely with the claudin-low and metaplastic breast cancer subtypes and correlates negatively with pathological complete response. Additionally, the expression level of FOXC1, another EMT inducer, correlates strongly with poor survival of breast cancer patients.

Core epithelial-to-mesenchymal transition interactome gene-expression signature is associated with claudin-low and metaplastic breast cancer subtypes

PubMed Central

Taube, Joseph H.; Herschkowitz, Jason I.; Komurov, Kakajan; Zhou, Alicia Y.; Gupta, Supriya; Yang, Jing; Hartwell, Kimberly; Onder, Tamer T.; Gupta, Piyush B.; Evans, Kurt W.; Hollier, Brett G.; Ram, Prahlad T.; Lander, Eric S.; Rosen, Jeffrey M.; Weinberg, Robert A.; Mani, Sendurai A.

2010-01-01

The epithelial-to-mesenchymal transition (EMT) produces cancer cells that are invasive, migratory, and exhibit stem cell characteristics, hallmarks of cells that have the potential to generate metastases. Inducers of the EMT include several transcription factors (TFs), such as Goosecoid, Snail, and Twist, as well as the secreted TGF-β1. Each of these factors is capable, on its own, of inducing an EMT in the human mammary epithelial (HMLE) cell line. However, the interactions between these regulators are poorly understood. Overexpression of each of the above EMT inducers up-regulates a subset of other EMT-inducing TFs, with Twist, Zeb1, Zeb2, TGF-β1, and FOXC2 being commonly induced. Up-regulation of Slug and FOXC2 by either Snail or Twist does not depend on TGF-β1 signaling. Gene expression signatures (GESs) derived by overexpressing EMT-inducing TFs reveal that the Twist GES and Snail GES are the most similar, although the Goosecoid GES is the least similar to the others. An EMT core signature was derived from the changes in gene expression shared by up-regulation of Gsc, Snail, Twist, and TGF-β1 and by down-regulation of E-cadherin, loss of which can also trigger an EMT in certain cell types. The EMT core signature associates closely with the claudin-low and metaplastic breast cancer subtypes and correlates negatively with pathological complete response. Additionally, the expression level of FOXC1, another EMT inducer, correlates strongly with poor survival of breast cancer patients. PMID:20713713

Conformational epitopes at cadherin calcium-binding sites and p120-catenin phosphorylation regulate cell adhesion

PubMed Central

Petrova, Yuliya I.; Spano, MarthaJoy M.; Gumbiner, Barry M.

2012-01-01

We investigated changes in cadherin structure at the cell surface that regulate its adhesive activity. Colo 205 cells are nonadhesive cells with a full but inactive complement of E-cadherin–catenin complexes at the cell surface, but they can be triggered to adhere and form monolayers. We were able to distinguish the inactive and active states of E-cadherin at the cell surface by using a special set of monoclonal antibodies (mAbs). Another set of mAbs binds E-cadherin and strongly activates adhesion. In other epithelial cell types these activating mAbs inhibit growth factor–induced down-regulation of adhesion and epithelial morphogenesis, indicating that these phenomena are also controlled by E-cadherin activity at the cell surface. Both types of mAbs recognize conformational epitopes at different interfaces between extracellular cadherin repeat domains (ECs), especially near calcium-binding sites. Activation also induces p120-catenin dephosphorylation, as well as changes in the cadherin cytoplasmic domain. Moreover, phospho-site mutations indicate that dephosphorylation of specific Ser/Thr residues in the N-terminal domain of p120-catenin mediate adhesion activation. Thus physiological regulation of the adhesive state of E-cadherin involves physical and/or conformational changes in the EC interface regions of the ectodomain at the cell surface that are mediated by catenin-associated changes across the membrane. PMID:22513089

Apico-basal forces exerted by apoptotic cells drive epithelium folding.

PubMed

Monier, Bruno; Gettings, Melanie; Gay, Guillaume; Mangeat, Thomas; Schott, Sonia; Guarner, Ana; Suzanne, Magali

2015-02-12

Epithelium folding is a basic morphogenetic event that is essential in transforming simple two-dimensional epithelial sheets into three-dimensional structures in both vertebrates and invertebrates. Folding has been shown to rely on apical constriction. The resulting cell-shape changes depend either on adherens junction basal shift or on a redistribution of myosin II, which could be driven by mechanical signals. Yet the initial cellular mechanisms that trigger and coordinate cell remodelling remain largely unknown. Here we unravel the active role of apoptotic cells in initiating morphogenesis, thus revealing a novel mechanism of epithelium folding. We show that, in a live developing tissue, apoptotic cells exert a transient pulling force upon the apical surface of the epithelium through a highly dynamic apico-basal myosin II cable. The apoptotic cells then induce a non-autonomous increase in tissue tension together with cortical myosin II apical stabilization in the surrounding tissue, eventually resulting in epithelium folding. Together our results, supported by a theoretical biophysical three-dimensional model, identify an apoptotic myosin-II-dependent signal as the initial signal leading to cell reorganization and tissue folding. This work further reveals that, far from being passively eliminated as generally assumed (for example, during digit individualization), apoptotic cells actively influence their surroundings and trigger tissue remodelling through regulation of tissue tension.

Lipophilic components of diesel exhaust particles induce pro-inflammatory responses in human endothelial cells through AhR dependent pathway(s).

PubMed

Brinchmann, Bendik C; Skuland, Tonje; Rambøl, Mia H; Szoke, Krisztina; Brinchmann, Jan E; Gutleb, Arno C; Moschini, Elisa; Kubátová, Alena; Kukowski, Klara; Le Ferrec, Eric; Lagadic-Gossmann, Dominique; Schwarze, Per E; Låg, Marit; Refsnes, Magne; Øvrevik, Johan; Holme, Jørn A

2018-05-11

Exposure to traffic-derived particulate matter (PM), such as diesel exhaust particles (DEP), is a leading environmental cause of cardiovascular disease (CVD), and may contribute to endothelial dysfunction and development of atherosclerosis. It is still debated how DEP and other inhaled PM can contribute to CVD. However, organic chemicals (OC) adhered to the particle surface, are considered central to many of the biological effects. In the present study, we have explored the ability of OC from DEP to reach the endothelium and trigger pro-inflammatory reactions, a central step on the path to atherosclerosis. Exposure-relevant concentrations of DEP (0.12 μg/cm 2 ) applied on the epithelial side of an alveolar 3D tri-culture, rapidly induced pro-inflammatory and aryl hydrocarbon receptor (AhR)-regulated genes in the basolateral endothelial cells. These effects seem to be due to soluble lipophilic constituents rather than particle translocation. Extractable organic material of DEP (DEP-EOM) was next fractionated with increasing polarity, chemically characterized, and examined for direct effects on pro-inflammatory and AhR-regulated genes in human microvascular endothelial (HMEC-1) cells and primary human endothelial cells (PHEC) from four healthy donors. Exposure-relevant concentrations of lipophilic DEP-EOM (0.15 μg/cm 2 ) induced low to moderate increases in IL-1α, IL-1β, COX2 and MMP-1 gene expression, and the MMP-1 secretion was increased. By contrast, the more polar EOM had negligible effects, even at higher concentrations. Use of pharmacological inhibitors indicated that AhR and protease-activated receptor-2 (PAR-2) were central in regulation of EOM-induced gene expression. Some effects also seemed to be attributed to redox-responses, at least at the highest exposure concentrations tested. Although the most lipophilic EOM, that contained the majority of PAHs and aliphatics, had the clearest low-concentration effects, there was no straight-forward link between chemical composition and biological effects. Lipophilic and semi-lipophilic chemicals seemed to detach from DEP, translocate through alveolar epithelial cells and trigger pro-inflammatory reactions in endothelial cells at exposure-relevant concentrations. These effects appeared to be triggered by AhR agonists, and involve PAR-2 signaling.

Arousal of cancer-associated stroma: overexpression of palladin activates fibroblasts to promote tumor invasion.

PubMed

Brentnall, Teresa A; Lai, Lisa A; Coleman, Joshua; Bronner, Mary P; Pan, Sheng; Chen, Ru

2012-01-01

Cancer-associated fibroblasts, comprised of activated fibroblasts or myofibroblasts, are found in the stroma surrounding solid tumors. These myofibroblasts promote invasion and metastasis of cancer cells. Mechanisms regulating the activation of the fibroblasts and the initiation of invasive tumorigenesis are of great interest. Upregulation of the cytoskeletal protein, palladin, has been detected in the stromal myofibroblasts surrounding many solid cancers and in expression screens for genes involved in invasion. Using a pancreatic cancer model, we investigated the functional consequence of overexpression of exogenous palladin in normal fibroblasts in vitro and its effect on the early stages of tumor invasion. Palladin expression in stromal fibroblasts occurs very early in tumorigenesis. In vivo, concordant expression of palladin and the myofibroblast marker, alpha smooth muscle actin (α-SMA), occurs early at the dysplastic stages in peri-tumoral stroma and progressively increases in pancreatic tumorigenesis. In vitro introduction of exogenous 90 kD palladin into normal human dermal fibroblasts (HDFs) induces activation of stromal fibroblasts into myofibroblasts as marked by induction of α-SMA and vimentin, and through the physical change of cell morphology. Moreover, palladin expression in the fibroblasts enhances cellular migration, invasion through the extracellular matrix, and creation of tunnels through which cancer cells can follow. The fibroblast invasion and creation of tunnels results from the development of invadopodia-like cellular protrusions which express invadopodia proteins and proteolytic enzymes. Palladin expression in fibroblasts is triggered by the co-culture of normal fibroblasts with k-ras-expressing epithelial cells. Overall, palladin expression can impart myofibroblast properties, in turn promoting the invasive potential of these peri-tumoral cells with invadopodia-driven degradation of extracellular matrix. Palladin expression in fibroblasts can be triggered by k-ras expression in adjacent epithelial cells. This data supports a model whereby palladin-activated fibroblasts facilitate stromal-dependent metastasis and outgrowth of tumorigenic epithelium.

CD103+ intraepithelial T cells in high-grade serous ovarian cancer are phenotypically diverse TCRαβ+ CD8αβ+ T cells that can be targeted for cancer immunotherapy

PubMed Central

Workel, Hagma H.; Tijans, Aline M.; Terwindt, Anouk L.J.; Brunekreeft, Kim L.; Plat, Annechien; Klip, Harry G.; Eggink, Florine A.; Leffers, Ninke; Helfrich, Wijnand; Samplonius, Douwe F.; Bremer, Edwin; Wisman, G. Bea A.; Daemen, Toos; Duiker, Evelien W.; Hollema, Harry; Nijman, Hans W.; de Bruyn, Marco

2016-01-01

CD103+ tumor-infiltrating lymphocytes (TIL) have been linked to specific epithelial infiltration and a prolonged survival in high-grade serous epithelial ovarian cancer (HGSC). However, whether these cells are induced as part of an ongoing anti-HGSC immune response or represent non-specifically expanded resident or mucosal lymphocytes remains largely unknown. In this study, we first confirmed that CD103+ TIL from HGSC were predominantly localized in the cancer epithelium and were strongly correlated with an improved prognosis. We further demonstrate that CD103+ TIL were almost exclusively CD3+ TCRαβ+ CD8αβ+ CD4- T cells, but heterogeneously expressed T cell memory and differentiation markers. Activation of peripheral T cells in the presence of HGSC was sufficient to trigger induction of CD103 in over 90% of all CD8+ cells in a T cell receptor (TCR)- and TGFβR1-dependent manner. Finally, CD103+ TIL isolated from primary HGSC showed signs of recent activation and dominantly co-expressed key immunotherapeutic targets PD-1 and CD27. Taken together, our data indicate CD103+ TIL in HGSC are formed as the result of an adaptive anti-tumor immune response that might be reactivated by (dual) checkpoint inhibition. PMID:27650547

SPHK1 (sphingosine kinase 1) induces epithelial-mesenchymal transition by promoting the autophagy-linked lysosomal degradation of CDH1/E-cadherin in hepatoma cells.

PubMed

Liu, Hong; Ma, Yan; He, Hong-Wei; Zhao, Wu-Li; Shao, Rong-Guang

2017-05-04

SPHK1 (sphingosine kinase 1), a regulator of sphingolipid metabolites, plays a causal role in the development of hepatocellular carcinoma (HCC) through augmenting HCC invasion and metastasis. However, the mechanism by which SPHK1 signaling promotes invasion and metastasis in HCC remains to be clarified. Here, we reported that SPHK1 induced the epithelial-mesenchymal transition (EMT) by accelerating CDH1/E-cadherin lysosomal degradation and facilitating the invasion and metastasis of HepG2 cells. Initially, we found that SPHK1 promoted cell migration and invasion and induced the EMT process through decreasing the expression of CDH1, which is an epithelial marker. Furthermore, SPHK1 accelerated the lysosomal degradation of CDH1 to induce EMT, which depended on TRAF2 (TNF receptor associated factor 2)-mediated macroautophagy/autophagy activation. In addition, the inhibition of autophagy recovered CDH1 expression and reduced cell migration and invasion through delaying the degradation of CDH1 in SPHK1-overexpressing cells. Moreover, the overexpression of SPHK1 produced intracellular sphingosine-1-phosphate (S1P). In response to S1P stimulation, TRAF2 bound to BECN1/Beclin 1 and catalyzed the lysine 63-linked ubiquitination of BECN1 for triggering autophagy. The deletion of the RING domain of TRAF2 inhibited autophagy and the interaction of BECN1 and TRAF2. Our findings define a novel mechanism responsible for the regulation of the EMT via SPHK1-TRAF2-BECN1-CDH1 signal cascades in HCC cells. Our work indicates that the blockage of SPHK1 activity to attenuate autophagy may be a promising strategy for the prevention and treatment of HCC.

Changes in the distribution of type II transmembrane serine protease, TMPRSS2 and in paracellular permeability in IPEC-J2 cells exposed to oxidative stress.

PubMed

Paszti-Gere, Erzsebet; Barna, Reka Fanni; Kovago, Csaba; Szauder, Ipoly; Ujhelyi, Gabriella; Jakab, Csaba; Meggyesházi, Nóra; Szekacs, Andras

2015-04-01

The effect of oxidative stress on barrier integrity and localization of transmembrane serine proteinase 2 (TMPRSS2) were studied using porcine epithelial IPEC-J2 cells on membrane inserts. Increased paracellular permeability of FITC-dextran 4 kDa (fluorescence intensity 43,508 ± 2,391 versus 3,550 ± 759) and that of gentamicin (3.41 ± 0.06 % increase to controls) were measured parallel with the reduced transepithelial electrical resistance (23.3 ± 4.06 % decrease) of cell layers 6 h after 1 h 1 mM H2O2 treatment. The immunohistochemical localization of adherens junctional β-catenin was not affected by reactive oxygen species (ROS) up to 4 mM H2O2. Peroxide-triggered enhanced paracellular permeability of IPEC-J2 cell layer was accompanied by predominantly cytoplasmic occurrence of TMPRSS2 embedded in cell membrane under physiological conditions. These results support that ROS can influence paracellular gate opening via multifaceted mode of action without involvement of β-catenin redistribution in adherens junction. Altered distribution pattern of TMPRSS2 and relocalized transmembrane serine protease activity may contribute to weakening of epithelial barrier integrity under acute oxidative stress.

Amniotic Epithelial Cells: A New Tool to Combat Aging and Age-Related Diseases?

PubMed Central

Di Germanio, Clara; Bernier, Michel; de Cabo, Rafael; Barboni, Barbara

2016-01-01

The number of elderly people is growing at an unprecedented rate and this increase of the aging population is expected to have a direct impact on the incidence of age-related diseases and healthcare-associated costs. Thus, it is imperative that new tools are developed to fight and slow age-related diseases. Regenerative medicine is a promising strategy for the maintenance of health and function late in life; however, stem cell-based therapies face several challenges including rejection and tumor transformation. As an alternative, the placenta offers an extraordinary source of fetal stem cells, including the amniotic epithelial cells (AECs), which retain some of the characteristics of embryonic stem cells, but show low immunogenicity, together with immunomodulatory and anti-inflammatory activities. Because of these characteristics, AECs have been widely utilized in regenerative medicine. This perspective highlights different mechanisms triggered by transplanted AECs that could be potentially useful for anti-aging therapies, which include: Graft and differentiation for tissue regeneration in age-related settings, anti-inflammatory behavior to combat “inflammaging,” anti-tumor activity, direct lifespan and healthspan extension properties, and possibly rejuvenation in a manner reminiscent of heterochronic parabiosis. Here, we critically discuss benefits and limitation of AECs-based therapies in age-related diseases. PMID:27921031

The notochord breaks bilateral symmetry by controlling cell shapes in the zebrafish laterality organ.

PubMed

Compagnon, Julien; Barone, Vanessa; Rajshekar, Srivarsha; Kottmeier, Rita; Pranjic-Ferscha, Kornelija; Behrndt, Martin; Heisenberg, Carl-Philipp

2014-12-22

Kupffer's vesicle (KV) is the zebrafish organ of laterality, patterning the embryo along its left-right (LR) axis. Regional differences in cell shape within the lumen-lining KV epithelium are essential for its LR patterning function. However, the processes by which KV cells acquire their characteristic shapes are largely unknown. Here, we show that the notochord induces regional differences in cell shape within KV by triggering extracellular matrix (ECM) accumulation adjacent to anterior-dorsal (AD) regions of KV. This localized ECM deposition restricts apical expansion of lumen-lining epithelial cells in AD regions of KV during lumen growth. Our study provides mechanistic insight into the processes by which KV translates global embryonic patterning into regional cell shape differences required for its LR symmetry-breaking function. Copyright © 2014 Elsevier Inc. All rights reserved.

Vitamin K3 induces antiproliferative effect in cervical epithelial cells transformed by HPV 16 (SiHa cells) through the increase in reactive oxygen species production.

PubMed

de Carvalho Scharf Santana, Natália; Lima, Natália Alves; Desoti, Vânia Cristina; Bidóia, Danielle Lazarin; de Souza Bonfim Mendonça, Patrícia; Ratti, Bianca Altrão; Nakamura, Tânia Ueda; Nakamura, Celso Vataru; Consolaro, Marcia Edilaine Lopes; Ximenes, Valdecir Farias; de Oliveira Silva, Sueli

2016-10-01

Cervical cancer is characterized as an important public health problem. According to latest estimates, cancer of the cervix is the fourth most common cancer among women. Due to its high prevalence, the search for new and efficient drugs to treat this infection is continuous. The progression of HPV-associated cervical cancer involves the expression of two viral proteins, E6 and E7, which are rapidly degraded by the ubiquitin-proteasome system through the increase in reactive oxygen species generation. Vitamins are essential to human substances, participate in the regulation of metabolism, and facilitate the process of energy transfer. Some early studies have indicated that vitamin K3 exerts antitumor activity by inducing cell death by apoptosis through an increase in the generation of reactive oxygen species. Thus, we evaluated the antiproliferative effect and a likely mechanism of action of vitamin K3 against cervical epithelial cells transformed by HPV 16 (SiHa cells) assessing the production of total ROS, the mitochondrial membrane potential, the cell morphology, the cell volume, and the cell membrane integrity. Our results show that vitamin K3 induces an increase in ROS production in SiHa cells, triggering biochemical and morphological events, such as depolarization of mitochondrial membrane potential and decreasing cell volume. Our data showed that vitamin K3 generates an oxidative imbalance in SiHa cells, leading to mechanisms that induce cell death by apoptosis.

Angiotensin-(1-7) counteracts the transforming effects triggered by angiotensin II in breast cancer cells.

PubMed

Cambados, Nadia; Walther, Thomas; Nahmod, Karen; Tocci, Johanna M; Rubinstein, Natalia; Böhme, Ilka; Simian, Marina; Sampayo, Rocío; Del Valle Suberbordes, Melisa; Kordon, Edith C; Schere-Levy, Carolina

2017-10-24

Angiotensin (Ang) II, the main effector peptide of the renin-angiotensin system, has been implicated in multiple aspects of cancer progression such as proliferation, migration, invasion, angiogenesis and metastasis. Ang-(1-7), is a biologically active heptapeptide, generated predominantly from AngII by the enzymatic activity of angiotensin converting enzyme 2. Previous studies have shown that Ang-(1-7) counterbalances AngII actions in different pathophysiological settings. In this study, we have analysed the impact of Ang-(1-7) on AngII-induced pro-tumorigenic features on normal murine mammary epithelial cells NMuMG and breast cancer cells MDA-MB-231. AngII stimulated the activation of the survival factor AKT in NMuMG cells mainly through the AT1 receptor. This PI3K/AKT pathway activation also promoted epithelial-mesenchymal transition (EMT). Concomitant treatment of NMuMG cells with AngII and Ang-(1-7) completely abolished EMT features induced by AngII. Furthermore, Ang-(1-7) abrogated AngII induced migration and invasion of the MDA-MB-231 cells as well as pro-angiogenic events such as the stimulation of MMP-9 activity and VEGF expression. Together, these results demonstrate for the first time that Ang-(1-7) counteracts tumor aggressive signals stimulated by AngII in breast cancer cells emerging the peptide as a potential therapy to prevent breast cancer progression.

Suppression of E-cadherin function drives the early stages of Ras-induced squamous cell carcinoma through up-regulation of FAK and Src

PubMed Central

Alt-Holland, Addy; Sowalsky, Adam; Szwec-Levin, Yonit; Shamis, Yulia; Hatch, Harold; Feig, Larry A.; Garlick, Jonathan A.

2011-01-01

Advanced stages of epithelial carcinogenesis involve the loss of intercellular adhesion, but it remains unclear how proteins that regulate alterations in cell-cell and cell-matrix adhesion are deregulated to promote the early stages of cancer development. To address this, a three-dimensional human tissue model that mimics the incipient stages of Squamous Cell Carcinoma (SCC) was used to study how E-cadherin suppression promotes tumor progression in Ras-expressing human keratinocytes. We found that E-cadherin suppression triggered elevated mRNA and protein expression levels of Focal Adhesion Kinase (FAK), and increased FAK and Src activities above the level seen in Ras-expressing E-cadherin-competent keratinocytes. sh-RNA-mediated depletion of FAK and Src restored E-cadherin expression levels by increasing its stability in the membrane, and blocked tumor cell invasion in tissues. Surface transplantation of these tissues to mice resulted in reversion of the tumor phenotype to low-grade tumor islands in contrast to control tissues that manifested an aggressive, high-grade SCC. These findings suggest that the tumor-promoting effect of E-cadherin suppression, a common event in SCC development, is exacerbated by enhanced E-cadherin degradation induced by elevated FAK and Src activities. Furthermore, they imply that targeting FAK or Src in human epithelial cells with neoplastic potential may inhibit the early stages of SCC. PMID:21716326

Resistance to Fluid Shear Stress Is a Conserved Biophysical Property of Malignant Cells

PubMed Central

Henry, Michael D.

2012-01-01

During metastasis, cancer cells enter the circulation in order to gain access to distant tissues, but how this fluid microenvironment influences cancer cell biology is poorly understood. A longstanding view is that circulating cancer cells derived from solid tissues may be susceptible to damage from hemodynamic shear forces, contributing to metastatic inefficiency. Here we report that compared to non-transformed epithelial cells, transformed cells are remarkably resistant to fluid shear stress (FSS) in a microfluidic protocol, exhibiting a biphasic decrease in viability when subjected to a series of millisecond pulses of high FSS. We show that magnitude of FSS resistance is influenced by several oncogenes, is an adaptive and transient response triggered by plasma membrane damage and requires extracellular calcium and actin cytoskeletal dynamics. This novel property of malignant cancer cells may facilitate hematogenous metastasis and indicates, contrary to expectations, that cancer cells are quite resistant to destruction by hemodynamic shear forces. PMID:23226552

Intestinal Epithelial Cell Response to Clostridium difficile Flagella.

PubMed

Batah, Jameel; Kansau, Imad

2016-01-01

Clostridium difficile is the bacterium responsible for most antibiotic-associated diarrhea in North America and Europe. This bacterium, which colonizes the gut of humans and animals, produces toxins that are known to contribute directly to damage of the gut. It is known that bacterial flagella are involved in intestinal lesions through the inflammatory host response. The C. difficile flagellin recognizes TLR5 and consequently activates the NF-κB and the MAPK signaling pathways which elicit the synthesis of pro-inflammatory cytokines. Increasing interest on the role of C. difficile flagella in eliciting this cell response was recently developed and the development of tools to study cell response triggered by C. difficile flagella will improve our understanding of the pathogenesis of C. difficile.

The enriched fraction of Vernonia cinerea L. induces apoptosis and inhibits multi-drug resistance transporters in human epithelial cancer cells.

PubMed

Appadath Beeran, Asmy; Maliyakkal, Naseer; Rao, Chamallamudi Mallikarjuna; Udupa, Nayanabhirama

2014-12-02

Vernonia cinerea Less. (VC) of the family Asteraceaes is considered as the sacred plant; 'Dasapushpam' which is ethnopharmacologically significant to the people of Kerala in India. In fact, VC has been used in the traditional system of medicine (Ayurveda) for the treatment of various ailments including cancer. Cytotoxicity of the ethanolic extract of VC (VC-ET), petroleum ether fraction (VC-PET), dichloromethane fraction (VC-DCM), n-butyl alcohol fraction (VC-BT), and rest fraction (VC-R) was evaluated in cervical carcinoma (HeLa), lung adenocarcinoma (A549), breast cancer (MCF-7), and colon carcinoma (Caco-2) cells using Sulforhodamine B (SRB) assay. The apoptotic effects of VC-DCM were assessed in cancer cells using Annexin V assay. The effects of VC-DCM on multi-drug resistance (MDR) transporters in HeLa, A549, MCF-7, and Caco-2 cells were evaluated using flow cytometry based functional assays. Similarly, drug uptake in cancer cells and sensitization of cancer cells towards chemotherapeutic drugs in the presence of VC-DCM were studied using Daunorubicin (DNR) accumulation assay and SRB assay, respectively. Cytotoxicity assay revealed that the enriched fraction of VC (VC-DCM) possessed dose-dependent cytotoxic effects in human epithelial cancer cells (HeLa, A549, MCF-7, and Caco-2). Further, treatment of cancer cells (HeLa, A549, MCF-7, and Caco-2) with VC-DCM led to a significant increase in both early and late apoptosis, indicating the induction of apoptosis. Interestingly, VC-DCM significantly inhibited functional activity of MDR transporters (ABC-B1 and ABC-G2), enhanced DNR-uptake in cancer cells, and sensitized cancer cells towards chemotherapeutic drug-mediated cytotoxicity, thus indicating the ability of VC-DCM to reverse MDR in cancer and enhance the cytotoxic effects of anticancer drugs. A methodological investigation on the anti-cancer properties of Vernonia cinerea Less. (VC) revealed that an enriched fraction of VC (VC-DCM) possessed cytotoxic effects, triggered apoptosis, inhibited MDR transporters, enhanced drug uptake, and sensitized cancer cells towards anticancer drug-mediated cytotoxicity in human epithelial cancer cells. Thus, VC appears to be promising for an effective treatment of various drug-resistant human epithelial cancers. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

PubMed Central

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

2002-01-01

The epithelial compartment of the human breast comprises two distinct lineages: the luminal epithelial and the myoepithelial lineage. We have shown previously that a subset of the luminal epithelial cells could convert to myoepithelial cells in culture signifying the possible existence of a progenitor cell. We therefore set out to identify and isolate the putative precursor in the luminal epithelial compartment. Using cell surface markers and immunomagnetic sorting, we isolated two luminal epithelial cell populations from primary cultures of reduction mammoplasties. The major population coexpresses sialomucin (MUC+) and epithelial-specific antigen (ESA+) whereas the minor population has a suprabasal position and expresses epithelial specific antigen but no sialomucin (MUC−/ESA+). Two cell lines were further established by transduction of the E6/E7 genes from human papilloma virus type 16. Both cell lines maintained a luminal epithelial phenotype as evidenced by expression of the tight junction proteins, claudin-1 and occludin, and by generation of a high transepithelial electrical resistance on semipermeable filters. Whereas in clonal cultures, the MUC+/ESA+ epithelial cell line was luminal epithelial restricted in its differentiation repertoire, the suprabasal-derived MUC−/ESA+ epithelial cell line was able to generate itself as well as MUC+/ESA+ epithelial cells and Thy-1+/α-smooth muscle actin+ (ASMA+) myoepithelial cells. The MUC−/ESA+ epithelial cell line further differed from the MUC+/ESA+ epithelial cell line by the expression of keratin K19, a feature of a subpopulation of epithelial cells in terminal duct lobular units in vivo. Within a reconstituted basement membrane, the MUC+/ESA+ epithelial cell line formed acinus-like spheres. In contrast, the MUC−/ESA+ epithelial cell line formed elaborate branching structures resembling uncultured terminal duct lobular units both by morphology and marker expression. Similar structures were obtained by inoculating the extracellular matrix-embedded cells subcutaneously in nude mice. Thus, MUC−/ESA+ epithelial cells within the luminal epithelial lineage may function as precursor cells of terminal duct lobular units in the human breast. PMID:11914275

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

PubMed Central

Yasen, Aizezi; Herrera, Rossana; Rosbe, Kristina

2017-01-01

Oropharyngeal mucosal epithelia of fetuses/neonates/infants and the genital epithelia of adults play a critical role in HIV-1 mother-to-child transmission and sexual transmission of virus, respectively. To study the mechanisms of HIV-1 transmission through mucosal epithelium, we established polarized tonsil, cervical and foreskin epithelial cells. Analysis of HIV-1 transmission through epithelial cells showed that approximately 0.05% of initially inoculated virions transmigrated via epithelium. More than 90% of internalized virions were sequestered in the endosomes of epithelial cells, including multivesicular bodies (MVBs) and vacuoles. Intraepithelial HIV-1 remained infectious for 9 days without viral release. Release of sequestered intraepithelial HIV-1 was induced by the calcium ionophore ionomycin and by cytochalasin D, which increase intracellular calcium and disrupt the cortical actin of epithelial cells, respectively. Cocultivation of epithelial cells containing HIV-1 with activated peripheral blood mononuclear cells and CD4+ T lymphocytes led to the disruption of epithelial cortical actin and spread of virus from epithelial cells to lymphocytes. Treatment of epithelial cells with proinflammatory cytokines tumor necrosis factor-alpha and interferon gamma also induced reorganization of cortical actin and release of virus. Inhibition of MVB formation by small interfering RNA (siRNA)-mediated silencing of its critical protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) expression reduced viral sequestration in epithelial cells and its transmission from epithelial cells to lymphocytes by ~60–70%. Furthermore, inhibition of vacuole formation of epithelial cells by siRNA-inactivated rabankyrin-5 expression also significantly reduced HIV-1 sequestration in epithelial cells and spread of virus from epithelial cells to lymphocytes. Interaction of the intercellular adhesion molecule-1 of epithelial cells with the function-associated antigen-1 of lymphocytes was important for inducing the release of sequestered HIV-1 from epithelial cells and facilitating cell-to-cell spread of virus from epithelial cells to lymphocytes. This mechanism may serve as a pathway of HIV-1 mucosal transmission. PMID:28241053

Nivalenol induces oxidative stress and increases deoxynivalenol pro-oxidant effect in intestinal epithelial cells

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

Del Regno, Marisanta; Adesso, Simona; Popolo, Ada

Mycotoxins are secondary fungal metabolites often found as contaminants in almost all agricultural commodities worldwide, and the consumption of food or feed contaminated by mycotoxins represents a major risk for human and animal health. Reactive oxygen species are normal products of cellular metabolism. However, disproportionate generation of reactive oxygen species poses a serious problem to bodily homeostasis and causes oxidative tissue damage. In this study we analyzed the effect of two trichothecenes mycotoxins: nivalenol and deoxynivalenol, alone and in combination, on oxidative stress in the non-tumorigenic intestinal epithelial cell line IEC-6. Our results indicate the pro-oxidant nivalenol effect in IEC-6,more » the stronger pro-oxidant effect of nivalenol when compared to deoxynivalenol and, interestingly, that nivalenol increases deoxynivalenol pro-oxidative effects. Mechanistic studies indicate that the observed effects were mediated by NADPH oxidase, calcium homeostasis alteration, NF-kB and Nrf2 pathways activation and by iNOS and nitrotyrosine formation. The toxicological interaction by nivalenol and deoxynivalenol reported in this study in IEC-6, points out the importance of the toxic effect of these mycotoxins, mostly in combination, further highlighting the risk assessment process of these toxins that are of growing concern. - Highlights: • Nivalenol induces oxidative stress in intestinal epithelial cells (IECs). • Nivalenol increases deoxynivalenol pro-oxidant effects in IECs. • Nivalenol and deoxynivalenol trigger antioxidant response IECs. • These results indicate the importance of mycotoxins co-contamination.« less

Expression of taste receptors in Solitary Chemosensory Cells of rodent airways

PubMed Central

2011-01-01

Background Chemical irritation of airway mucosa elicits a variety of reflex responses such as coughing, apnea, and laryngeal closure. Inhaled irritants can activate either chemosensitive free nerve endings, laryngeal taste buds or solitary chemosensory cells (SCCs). The SCC population lies in the nasal respiratory epithelium, vomeronasal organ, and larynx, as well as deeper in the airway. The objective of this study is to map the distribution of SCCs within the airways and to determine the elements of the chemosensory transduction cascade expressed in these SCCs. Methods We utilized a combination of immunohistochemistry and molecular techniques (rtPCR and in situ hybridization) on rats and transgenic mice where the Tas1R3 or TRPM5 promoter drives expression of green fluorescent protein (GFP). Results Epithelial SCCs specialized for chemoreception are distributed throughout much of the respiratory tree of rodents. These cells express elements of the taste transduction cascade, including Tas1R and Tas2R receptor molecules, α-gustducin, PLCβ2 and TrpM5. The Tas2R bitter taste receptors are present throughout the entire respiratory tract. In contrast, the Tas1R sweet/umami taste receptors are expressed by numerous SCCs in the nasal cavity, but decrease in prevalence in the trachea, and are absent in the lower airways. Conclusions Elements of the taste transduction cascade including taste receptors are expressed by SCCs distributed throughout the airways. In the nasal cavity, SCCs, expressing Tas1R and Tas2R taste receptors, mediate detection of irritants and foreign substances which trigger trigeminally-mediated protective airway reflexes. Lower in the respiratory tract, similar chemosensory cells are not related to the trigeminal nerve but may still trigger local epithelial responses to irritants. In total, SCCs should be considered chemoreceptor cells that help in preventing damage to the respiratory tract caused by inhaled irritants and pathogens. PMID:21232137

Expression of taste receptors in solitary chemosensory cells of rodent airways.

PubMed

Tizzano, Marco; Cristofoletti, Mirko; Sbarbati, Andrea; Finger, Thomas E

2011-01-13

Chemical irritation of airway mucosa elicits a variety of reflex responses such as coughing, apnea, and laryngeal closure. Inhaled irritants can activate either chemosensitive free nerve endings, laryngeal taste buds or solitary chemosensory cells (SCCs). The SCC population lies in the nasal respiratory epithelium, vomeronasal organ, and larynx, as well as deeper in the airway. The objective of this study is to map the distribution of SCCs within the airways and to determine the elements of the chemosensory transduction cascade expressed in these SCCs. We utilized a combination of immunohistochemistry and molecular techniques (rtPCR and in situ hybridization) on rats and transgenic mice where the Tas1R3 or TRPM5 promoter drives expression of green fluorescent protein (GFP). Epithelial SCCs specialized for chemoreception are distributed throughout much of the respiratory tree of rodents. These cells express elements of the taste transduction cascade, including Tas1R and Tas2R receptor molecules, α-gustducin, PLCβ2 and TrpM5. The Tas2R bitter taste receptors are present throughout the entire respiratory tract. In contrast, the Tas1R sweet/umami taste receptors are expressed by numerous SCCs in the nasal cavity, but decrease in prevalence in the trachea, and are absent in the lower airways. Elements of the taste transduction cascade including taste receptors are expressed by SCCs distributed throughout the airways. In the nasal cavity, SCCs, expressing Tas1R and Tas2R taste receptors, mediate detection of irritants and foreign substances which trigger trigeminally-mediated protective airway reflexes. Lower in the respiratory tract, similar chemosensory cells are not related to the trigeminal nerve but may still trigger local epithelial responses to irritants. In total, SCCs should be considered chemoreceptor cells that help in preventing damage to the respiratory tract caused by inhaled irritants and pathogens.

Salmonella modulation of host cell gene expression promotes its intracellular growth.

PubMed

Hannemann, Sebastian; Gao, Beile; Galán, Jorge E

2013-01-01

Salmonella Typhimurium has evolved a complex functional interface with its host cell largely determined by two type III secretion systems (T3SS), which through the delivery of bacterial effector proteins modulate a variety of cellular processes. We show here that Salmonella Typhimurium infection of epithelial cells results in a profound transcriptional reprogramming that changes over time. This response is triggered by Salmonella T3SS effector proteins, which stimulate unique signal transduction pathways leading to STAT3 activation. We found that the Salmonella-stimulated changes in host cell gene expression are required for the formation of its specialized vesicular compartment that is permissive for its intracellular replication. This study uncovers a cell-autonomous process required for Salmonella pathogenesis potentially opening up new avenues for the development of anti-infective strategies that target relevant host pathways.

Identification of embryonic precursor cells that differentiate into thymic epithelial cells expressing autoimmune regulator

PubMed Central

Takizawa, Nobukazu; Miyauchi, Maki; Yanai, Hiromi; Tateishi, Ryosuke; Shinzawa, Miho; Yoshinaga, Riko; Kurihara, Masaaki; Yasuda, Hisataka; Sakamoto, Reiko; Yoshida, Nobuaki

2016-01-01

Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire+ mTECs) is unclear. Here, we describe novel embryonic precursors of Aire+ mTECs. We found the candidate precursors of Aire+ mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire+ mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire+ mTECs and efficiently suppressed the onset of autoimmunity induced by Aire+ mTEC deficiency. Mechanistically, pMECs differentiated into Aire+ mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-β receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire+ mTECs. PMID:27401343

Taurine protects HK-2 cells from oxidized LDL-induced cytotoxicity via the ROS-mediated mitochondrial and p53-related apoptotic pathways.

PubMed

Chang, Chun-Yu; Shen, Chao-Yu; Kang, Chao-Kai; Sher, Yuh-Pyng; Sheu, Wayne H-H; Chang, Chia-Che; Lee, Tsung-Han

2014-09-15

Oxidized LDL (oxLDL) induces a pro-oxidative environment and promotes apoptosis, causing the progression of renal diseases in humans. Taurine is a semi-essential amino acid in mammals and has been shown to be a potent endogenous antioxidant. The kidney plays a pivotal role in maintaining the balance of taurine. However, the mechanisms underlying the protective effects of taurine against oxLDL-induced injury in renal epithelial cells have not been clarified. In the present study, we investigated the anti-apoptotic effects of taurine on human proximal tubular epithelial (HK-2) cells exposed to oxLDL and explored the related mechanisms. We observed that oxLDL increased the contents of ROS and of malondialdehyde (MDA), which is a lipid peroxidation by-product that acts as an indicator of the cellular oxidation status. In addition, oxLDL induced cell death and apoptosis in HK-2 cells. Pretreatment with taurine at 100 μM significantly attenuated the oxLDL-induced cytotoxicity. We determined that oxLDL triggered the phosphorylation of ERK and, in turn, the activation of p53 and other apoptosis-related events, including calcium accumulation, destabilization of the mitochondrial permeability and disruption of the balance between pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins. The malfunctions induced by oxLDL were effectively blocked by taurine. Thus, our results suggested that taurine exhibits potential therapeutic activity by preventing oxLDL-induced nephrotoxicity. The inhibition of oxLDL-induced epithelial apoptosis by taurine was at least partially due to its anti-oxidant activity and its ability to modulate the ERK and p53 apoptotic pathways. Copyright © 2014 Elsevier Inc. All rights reserved.

Metalloproteinase-dependent transforming growth factor-alpha release mediates neurotensin-stimulated MAP kinase activation in human colonic epithelial cells.

PubMed

Zhao, Dezheng; Zhan, Yanai; Koon, Hon Wai; Zeng, Huiyan; Keates, Sarah; Moyer, Mary P; Pothoulakis, Charalabos

2004-10-15

Expression of the neuropeptide neurotensin (NT) and its high affinity receptor (NTR1) is increased during the course of Clostridium difficile toxin A-induced acute colitis, and NTR1 antagonism attenuates the severity of toxin A-induced inflammation. We recently demonstrated in non-transformed human colonic epithelial NCM460 cells that NT treatment caused activation of a Ras-mediated MAP kinase pathway that significantly contributes to NT-induced interleukin-8 (IL-8) secretion. Here we used NCM460 cells, which normally express low levels of NTR1, and NCM460 cells stably transfected with NTR1 to identify the upstream signaling molecules involved in NT-NTR1-mediated MAP kinase activation. We found that inhibition of the epidermal growth factor receptor (EGFR) by either an EGFR neutralizing antibody or by its specific inhibitor AG1478 (0.2 microm) blocked NT-induced MAP kinase activation. Moreover, NT stimulated tyrosine phosphorylation of the EGFR, and pretreatment with a broad spectrum metalloproteinase inhibitor batimastat reduced NT-induced MAP kinase activation. Using neutralizing antibodies against the EGFR ligands EGF, heparin-binding-EGF, transforming growth factor-alpha (TGFalpha), or amphiregulin we have shown that only the anti-TGFalpha antibody significantly decreases NT-induced phosphorylation of EGFR and MAP kinases. Furthermore, inhibition of the EGF receptor by AG1478 significantly reduced NT-induced IL-8 promoter activity and IL-8 secretion. This is the first report demonstrating that NT binding to NTR1 transactivates the EGFR and that this response is linked to NT-mediated proinflammatory signaling. Our findings indicate that matrix metalloproteinase-mediated release of TGFalpha and subsequent EGFR transactivation triggers a NT-mediated MAP kinase pathway that leads to IL-8 gene expression in human colonic epithelial cells.

MicroRNA-9 enhances sensitivity to cetuximab in epithelial phenotype hepatocellular carcinoma cells through regulation of the eukaryotic translation initiation factor 5A-2.

PubMed

Xue, Fei; Liang, Yuntian; Li, Zhenrong; Liu, Yanhui; Zhang, Hongwei; Wen, Yu; Yan, Lei; Tang, Qiang; Xiao, Erhui; Zhang, Dongyi

2018-01-01

Hepatocellular carcinoma (HCC) is one of the most widespread malignant human tumors worldwide. Treatment options include radiotherapy, surgical intervention and chemotherapy; however, drug resistance is an ongoing treatment concern. In the present study, the effects of a microRNA (miR/miRNA), miR-9, on the sensitivity of HCC cell lines to the epidermal growth factor receptor inhibitor, cetuximab, were examined. miR-9 has been proposed to serve a role in tumorigenesis and tumor progression. In the present study, bioinformatics analyses identified the eukaryotic translation initiation factor 5A2 (eIF-5A-2) as a target of miR-9. The expression levels of miR-9 and eIF-5A-2 were examined by reverse transcription-quantitative polymerase chain reaction and HCC cell lines were transfected with miR-9 mimics and inhibitors to determine the effects of the miRNA on cell proliferation and viability. The miR-9 mimic was revealed to significantly increase the sensitivity of epithelial phenotype HCC cells (Hep3B and Huh7) to cetuximab, while the miR-9 inhibitor triggered the opposite effect. There were no significant differences in sensitivity to cetuximab observed in mesenchymal phenotype HCC cells (SNU387 and SNU449). Cells lines displaying high expression levels of eIF-5A-2 were more resistant to cetuximab. Transfection of cells with a miR-9 mimic resulted in downregulation of the expression of eIF-5A-2 mRNA, while an miR-9 inhibitor increased expression. When expression of eIF-5A-2 was knocked down with siRNA, the effects of miR-9 on cetuximab sensitivity were no longer observed. Taken together, these data support a role for miR-9 in enhancing the sensitivity of epithelial phenotype HCC cells to cetuximab through regulation of eIF-5A-2.

Designing 3-Dimensional In Vitro Oviduct Culture Systems to Study Mammalian Fertilization and Embryo Production.

PubMed

Ferraz, Marcia A M M; Henning, Heiko H W; Stout, Tom A E; Vos, Peter L A M; Gadella, Bart M

2017-07-01

The oviduct was long considered a largely passive conduit for gametes and embryos. However, an increasing number of studies into oviduct physiology have demonstrated that it specifically and significantly influences gamete interaction, fertilization and early embryo development. While oviduct epithelial cell (OEC) function has been examined during maintenance in conventional tissue culture dishes, cells seeded into these two-dimensional (2-D) conditions suffer a rapid loss of differentiated OEC characteristics, such as ciliation and secretory activity. Recently, three-dimensional (3-D) cell culture systems have been developed that make use of cell inserts to create basolateral and apical medium compartments with a confluent epithelial cell layer at the interface. Using such 3-D culture systems, OECs can be triggered to redevelop typical differentiated cell properties and levels of tissue organization can be developed that are not possible in a 2-D culture. 3-D culture systems can be further refined using new micro-engineering techniques (including microfluidics and 3-D printing) which can be used to produce 'organs-on-chips', i.e. live 3-D cultures that bio-mimic the oviduct. In this review, concepts for designing bio-mimic 3-D oviduct cultures are presented. The increased possibilities and concomitant challenges when trying to more closely investigate oviduct physiology, gamete activation, fertilization and embryo production are discussed.

Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2

PubMed Central

Li, Qiong; Kumar, Ashok; Gui, Jian-Fang; Yu, Fu-Shin X.

2008-01-01

Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-κB, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-κB activation in HUCL cells after lipoprotein lipase treatment and in cell lines expressing TLR4 or TLR9, but TLR2, indicating lipoprotein nature of the extracts. saLP induced the up-regulation of a variety of inflammatory cytokines and chemokines (IL-6, IL-8, ICAM-1) and antimicrobial molecules (hBD-2, LL-37, and iNOS), and homeostasis genes (Mn-SOD) at both the mRNA level and protein level. Similar inflammatory response to saLP was also observed in primarily cultured HCECs using the production of IL-6 as readout. Moreover, TLR2 neutralizing antibody blocked the saLP-induced secretion of IL-6, IL-8 and hBD2 in HUCL cells. Our findings suggest that saLP activates TLR2 and contributes to innate immune response in the cornea to S. aureus infection via production of proinflammatory cytokines and defense molecules. PMID:18191935

In vitro effects of anthocyanidins on sinonasal epithelial nitric oxide production and bacterial physiology

PubMed Central

Hariri, Benjamin M.; Payne, Sakeena J.; Chen, Bei; Mansfield, Corrine; Doghramji, Laurel J.; Adappa, Nithin D.; Palmer, James N.; Kennedy, David W.; Niv, Masha Y.

2016-01-01

Background: T2R bitter taste receptors play a crucial role in sinonasal innate immunity by upregulating mucociliary clearance and nitric oxide (NO) production in response to bitter gram-negative quorum-sensing molecules in the airway surface liquid. Previous studies showed that phytochemical flavonoid metabolites, known as anthocyanidins, taste bitter and have antibacterial effects. Our objectives were to examine the effects of anthocyanidins on NO production by human sinonasal epithelial cells and ciliary beat frequency, and their impact on common sinonasal pathogens Pseudomonas aeruginosa and Staphylococcus aureus. Methods: Ciliary beat frequency and NO production were measured by using digital imaging of differentiated air-liquid interface cultures prepared from primary human cells isolated from residual surgical material. Plate-based assays were used to determine the effects of anthocyanidins on bacterial swimming and swarming motility. Biofilm formation and planktonic growth were also assessed. Results: Anthocyanidin compounds triggered epithelial cells to produce NO but not through T2R receptors. However, anthocyanidins did not impact ciliary beat frequency. Furthermore, they did not reduce biofilm formation or planktonic growth of P. aeruginosa. In S. aureus, they did not reduce planktonic growth, and only one compound had minimal antibiofilm effects. The anthocyanidin delphinidin and anthocyanin keracyanin were found to promote bacterial swimming, whereas anthocyanidin cyanidin and flavonoid myricetin did not. No compounds that were tested inhibited bacterial swarming. Conclusion: Results of this study indicated that, although anthocyanidins may elicited an innate immune NO response from human cells, they do not cause an increase in ciliary beating and they may also cause a pathogenicity-enhancing effect in P. aeruginosa. Additional studies are necessary to understand how this would affect the use of anthocyanidins as therapeutics. This study emphasized the usefulness of in vitro screening of candidate compounds against multiple parameters of both epithelial and bacterial physiologies to prioritize candidates for in vivo therapeutic testing. PMID:27456596

P2X7 receptors regulate multiple types of membrane trafficking responses and non-classical secretion pathways.

PubMed

Qu, Yan; Dubyak, George R

2009-06-01

Activation of the P2X7 receptor (P2X7R) triggers a remarkably diverse array of membrane trafficking responses in leukocytes and epithelial cells. These responses result in altered profiles of cell surface lipid and protein composition that can modulate the direct interactions of P2X7R-expressing cells with other cell types in the circulation, in blood vessels, at epithelial barriers, or within sites of immune and inflammatory activation. Additionally, these responses can result in the release of bioactive proteins, lipids, and large membrane complexes into extracellular compartments for remote communication between P2X7R-expressing cells and other cells that amplify or modulate inflammation, immunity, and responses to tissue damages. This review will discuss P2X7R-mediated effects on membrane composition and trafficking in the plasma membrane (PM) and intracellular organelles, as well as actions of P2X7R in controlling various modes of non-classical secretion. It will review P2X7R regulation of: (1) phosphatidylserine distribution in the PM outer leaflet; (2) shedding of PM surface proteins; (3) release of PM-derived microvesicles or microparticles; (4) PM blebbing; (5) cell-cell fusion resulting in formation of multinucleate cells; (6) phagosome maturation and fusion with lysosomes; (7) permeability of endosomes with internalized pathogen-associated molecular patterns; (8) permeability/integrity of mitochondria; (9) exocytosis of secretory lysosomes; and (10) release of exosomes from multivesicular bodies.

Camel urine components display anti-cancer properties in vitro.

PubMed

Al-Yousef, Nujoud; Gaafar, Ameera; Al-Otaibi, Basem; Al-Jammaz, Ibrahim; Al-Hussein, Khaled; Aboussekhra, Abdelilah

2012-10-11

While camel urine (CU) is widely used in the Arabian Peninsula to treat various diseases, including cancer, its exact mechanism of action is still not defined. The objective of the present study is to investigate whether camel urine has anti-cancer effect on human cells in vitro. The annexinV/PI assay was used to assess apoptosis, and immunoblotting analysis determined the effect of CU on different apoptotic and oncogenic proteins. Furthermore, flow cytometry and Elispot were utilized to investigate cytotoxicity and the effect on the cell cycle as well as the production of cytokines, respectively. Camel urine showed cytotoxicity against various, but not all, human cancer cell lines, with only marginal effect on non-tumorigenic epithelial and normal fibroblast cells epithelial and fibroblast cells. Interestingly, 216 mg/ml of lyophilized CU inhibited cell proliferation and triggered more than 80% of apoptosis in different cancer cells, including breast carcinomas and medulloblastomas. Apoptosis was induced in these cells through the intrinsic pathway via Bcl-2 decrease. Furthermore, CU down-regulated the cancer-promoting proteins survivin, β-catenin and cyclin D1 and increased the level of the cyclin-dependent kinase inhibitor p21. In addition, we have shown that CU has no cytotoxic effect against peripheral blood mononuclear cells and has strong immuno-inducer activity through inducing IFN-γ and inhibiting the Th2 cytokines IL-4, IL-6 and IL-10. CU has specific and efficient anti-cancer and potent immune-modulator properties in vitro. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Prolyl isomerase Pin1 acts downstream of miR-200 to promote cancer stem-like cell traits in breast cancer

PubMed Central

Luo, Man-Li; Gong, Chang; Chen, Chun-Hau; Lee, Daniel Y.; Hu, Hai; Huang, Pengyu; Yao, Yandan; Guo, Wenjun; Reinhardt, Ferenc; Wulf, Gerburg; Lieberman, Judy; Zhou, Xiao Zhen; Song, Erwei; Lu, Kun Ping

2014-01-01

Breast cancer stem-like cells (BCSC) have been implicated in tumor growth, metastasis, drug resistance and relapse but druggable targets in appropriate subsets of this cell population have yet to be identified. Here we identify a fundamental role for the prolyl isomerase Pin1 in driving BCSC expansion, invasiveness and tumorigenicity, defining it as a key target of miR-200c which is known to be a critical regluator in BSCS. Pin1 overexpression expanded the growth and tumorigenicity of BCSC and triggered epithelial-mesenchymal transition (EMT). Conversely, genetic or pharmaacological inhibition of Pin1 reduced the abundance and self-renewal activity of BCSC. Moreover, moderate overexpression of miR-200c-resistant Pin1 rescued the BCSC defect in miR-200c-expressing cells. Genetic deletion of Pin1 also decreased the abundance and repopulating capability of normal mouse mammary stem cells. In human cells freshly isolated from reduction mammoplasty tissues, Pin1 overexpression endowed BCSC traits to normal breast epithelial cells, expanding both luminal and basal/myoepithelial lineages in these cells. In contrast, Pin1 silencing in primary breast cancer cells isolated from clinical samples inhibited the expansion, self-renewal activity and tumorigenesis of BCSC in vitro and in vivo. Overall, our work demonstrated that Pin1 is a pivotal regulator acting downstream of miR-200c to drive BCSC and breast tumorigenicity, highlighting a new therapeutic target to eradicate BCSC. PMID:24786790

Hexavalent chromium, a lung carcinogen, confers resistance to thermal stress and interferes with heat shock protein expression in human bronchial epithelial cells.

PubMed

Abreu, Patrícia L; Cunha-Oliveira, Teresa; Ferreira, Leonardo M R; Urbano, Ana M

2018-03-16

Exposure to hexavalent chromium [Cr(VI)], a lung carcinogen, triggers several types of cellular stresses, namely oxidative, genotoxic and proteotoxic stresses. Given the evolutionary character of carcinogenesis, it is tempting to speculate that cells that survive the stresses produced by this carcinogen become more resistant to subsequent stresses, namely those encountered during neoplastic transformation. To test this hypothesis, we determined whether pre-incubation with Cr(VI) increased the resistance of human bronchial epithelial cells (BEAS-2B cells) to the antiproliferative action of acute thermal shock, used here as a model for stress. In line with the proposed hypothesis, it was observed that, at mildly cytotoxic concentrations, Cr(VI) attenuated the antiproliferative effects of both cold and heat shock. Mechanistically, Cr(VI) interfered with the expression of two components of the stress response pathway: heat shock proteins Hsp72 and Hsp90α. Specifically, Cr(VI) significantly depleted the mRNA levels of the former and the protein levels of the latter. Significantly, these two proteins are members of heat shock protein (Hsp) families (Hsp70 and Hsp90, respectively) that have been implicated in carcinogenesis. Thus, our results confirm and extend previous studies showing the capacity of Cr(VI) to interfere with the expression of stress response components.

The differential role of human macrophage in triggering secondary bystander effects after either gamma-ray or carbon beam irradiation

PubMed Central

Dong, Chen; He, Mingyuan; Tu, Wenzhi; Konishi, Teruaki; Liu, Weili; Xie, Yuexia; Dang, Bingrong; Li, Wenjian; Uchihori, Yukio; Hei, Tom K.; Shao, Chunlin

2015-01-01

The abscopal effect could be an underlying factor in evaluating prognosis of radiotherapy. This study established an in vitro system to examine whether tumor-generated bystander signals could be transmitted by macrophages to further trigger secondary cellular responses after different irradiations, where human lung cancer NCI-H446 cells were irradiated with either γ-rays or carbon ions and co-cultured with human macrophage U937 cells, then these U937 cells were used as a bystander signal transmitter and co-cultured with human bronchial epithelial cells BEAS-2B. Results showed that U937 cells were only activated by γ-irradiated NCI-H446 cells so that the secondary injuries in BEAS-2B cells under carbon ion irradiation were weaker than γ-rays. Both TNF-α and IL-1α were involved in γ-irradiation induced secondary bystander effect but only TNF-α contributed to the carbon ion induced response. Further assay disclosed that IL-1α but not TNF-α was largely responsible for the activation of macrophages and the formation of micronucleus in BEAS-2B cells. These data suggest that macrophages could transfer secondary bystander signals and play a key role in the secondary bystander effect of photon irradiation while carbon ion irradiation has conspicuous advantage due to its reduced secondary injury. PMID:25896631

The differential role of human macrophage in triggering secondary bystander effects after either gamma-ray or carbon beam irradiation.

PubMed

Dong, Chen; He, Mingyuan; Tu, Wenzhi; Konishi, Teruaki; Liu, Weili; Xie, Yuexia; Dang, Bingrong; Li, Wenjian; Uchihori, Yukio; Hei, Tom K; Shao, Chunlin

2015-07-10

The abscopal effect could be an underlying factor in evaluating prognosis of radiotherapy. This study established an in vitro system to examine whether tumor-generated bystander signals could be transmitted by macrophages to further trigger secondary cellular responses after different irradiations, where human lung cancer NCI-H446 cells were irradiated with either γ-rays or carbon ions and co-cultured with human macrophage U937 cells, then these U937 cells were used as a bystander signal transmitter and co-cultured with human bronchial epithelial cells BEAS-2B. Results showed that U937 cells were only activated by γ-irradiated NCI-H446 cells so that the secondary injuries in BEAS-2B cells under carbon ion irradiation were weaker than γ-rays. Both TNF-α and IL-1α were involved in the γ-irradiation induced secondary bystander effect but only TNF-α contributed to the carbon ion induced response. Further assay disclosed that IL-1α but not TNF-α was largely responsible for the activation of macrophages and the formation of micronucleus in BEAS-2B cells. These data suggest that macrophages could transfer secondary bystander signals and play a key role in the secondary bystander effect of photon irradiation, while carbon ion irradiation has conspicuous advantage due to its reduced secondary injury. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Regulation of Telomere Homeostasis during Epstein-Barr virus Infection and Immortalization.

PubMed

Kamranvar, Siamak A; Masucci, Maria G

2017-08-09

The acquisition of unlimited proliferative potential is dependent on the activation of mechanisms for telomere maintenance, which counteracts telomere shortening and the consequent triggering of the DNA damage response, cell cycle arrest, and apoptosis. The capacity of Epstein Barr virus (EBV) to infect B-lymphocytes in vitro and transform the infected cells into autonomously proliferating immortal cell lines underlies the association of this human gamma-herpesvirus with a broad variety of lymphoid and epithelial cell malignancies. Current evidence suggests that both telomerase-dependent and -independent pathways of telomere elongation are activated in the infected cells during the early and late phases of virus-induced immortalization. Here we review the interaction of EBV with different components of the telomere maintenance machinery and the mechanisms by which the virus regulates telomere homeostasis in proliferating cells. We also discuss how these viral strategies may contribute to malignant transformation.

Listeria monocytogenes switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells.

PubMed

Kortebi, Mounia; Milohanic, Eliane; Mitchell, Gabriel; Péchoux, Christine; Prevost, Marie-Christine; Cossart, Pascale; Bierne, Hélène

2017-11-01

Listeria monocytogenes causes listeriosis, a foodborne disease that poses serious risks to fetuses, newborns and immunocompromised adults. This intracellular bacterial pathogen proliferates in the host cytosol and exploits the host actin polymerization machinery to spread from cell-to-cell and disseminate in the host. Here, we report that during several days of infection in human hepatocytes or trophoblast cells, L. monocytogenes switches from this active motile lifestyle to a stage of persistence in vacuoles. Upon intercellular spread, bacteria gradually stopped producing the actin-nucleating protein ActA and became trapped in lysosome-like vacuoles termed Listeria-Containing Vacuoles (LisCVs). Subpopulations of bacteria resisted degradation in LisCVs and entered a slow/non-replicative state. During the subculture of host cells harboring LisCVs, bacteria showed a capacity to cycle between the vacuolar and the actin-based motility stages. When ActA was absent, such as in ΔactA mutants, vacuolar bacteria parasitized host cells in the so-called "viable but non-culturable" state (VBNC), preventing their detection by conventional colony counting methods. The exposure of infected cells to high doses of gentamicin did not trigger the formation of LisCVs, but selected for vacuolar and VBNC bacteria. Together, these results reveal the ability of L. monocytogenes to enter a persistent state in a subset of epithelial cells, which may favor the asymptomatic carriage of this pathogen, lengthen the incubation period of listeriosis, and promote bacterial survival during antibiotic therapy.

Listeria monocytogenes switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells

PubMed Central

Mitchell, Gabriel

2017-01-01

Listeria monocytogenes causes listeriosis, a foodborne disease that poses serious risks to fetuses, newborns and immunocompromised adults. This intracellular bacterial pathogen proliferates in the host cytosol and exploits the host actin polymerization machinery to spread from cell-to-cell and disseminate in the host. Here, we report that during several days of infection in human hepatocytes or trophoblast cells, L. monocytogenes switches from this active motile lifestyle to a stage of persistence in vacuoles. Upon intercellular spread, bacteria gradually stopped producing the actin-nucleating protein ActA and became trapped in lysosome-like vacuoles termed Listeria-Containing Vacuoles (LisCVs). Subpopulations of bacteria resisted degradation in LisCVs and entered a slow/non-replicative state. During the subculture of host cells harboring LisCVs, bacteria showed a capacity to cycle between the vacuolar and the actin-based motility stages. When ActA was absent, such as in ΔactA mutants, vacuolar bacteria parasitized host cells in the so-called “viable but non-culturable” state (VBNC), preventing their detection by conventional colony counting methods. The exposure of infected cells to high doses of gentamicin did not trigger the formation of LisCVs, but selected for vacuolar and VBNC bacteria. Together, these results reveal the ability of L. monocytogenes to enter a persistent state in a subset of epithelial cells, which may favor the asymptomatic carriage of this pathogen, lengthen the incubation period of listeriosis, and promote bacterial survival during antibiotic therapy. PMID:29190284

Epithelial to mesenchymal transition-related proteins ZEB1, β-catenin, and β-tubulin-III in idiopathic pulmonary fibrosis.

PubMed

Chilosi, Marco; Caliò, Anna; Rossi, Andrea; Gilioli, Eliana; Pedica, Federica; Montagna, Licia; Pedron, Serena; Confalonieri, Marco; Doglioni, Claudio; Ziesche, Rolf; Grubinger, Markus; Mikulits, Wolfgang; Poletti, Venerino

2017-01-01

Epithelial to mesenchymal transition has been suggested as a relevant contributor to pulmonary fibrosis, but how and where this complex process is triggered in idiopathic pulmonary fibrosis is not fully understood. Beta-tubulin-III (Tubβ3), ZEB1, and β-catenin are partially under the negative control of miR-200, a family of micro-RNAs playing a major role in epithelial to mesenchymal transition, that are reduced in experimental lung fibrosis and idiopathic pulmonary fibrosis. We wonder whether in situ expression of these proteins is increased in idiopathic pulmonary fibrosis, to better understand the significance of miR-200 feedback loop and epithelial to mesenchymal transition. We investigated the immunohistochemical and immunofluorescent expression and precise location of ZEB1, Tubβ3, and β-catenin in tissue samples from 34 idiopathic pulmonary fibrosis cases and 21 controls (5 normal lungs and 16 other interstitial lung diseases). In 100% idiopathic pulmonary fibrosis samples, the three proteins were concurrently expressed in fibroblastic foci, as well in damaged epithelial cells overlying these lesions and in pericytes within neo-angiogenesis areas. These results were also confirmed by immunofluorescence assay. In controls the abnormal expression of the three proteins was absent or limited. This is the first study that relates concurrent expression of Tubβ3, ZEB1, and β-catenin to abnormal epithelial and myofibroblast differentiation in idiopathic pulmonary fibrosis, providing indirect but robust evidence of miR-200 deregulation and epithelial to mesenchymal transition activation in idiopathic pulmonary fibrosis. The abnormal expression and localization of these proteins in bronchiolar fibro-proliferative lesions are unique for idiopathic pulmonary fibrosis, and might represent a disease-specific marker in challenging lung biopsies.

Stress-induced premature senescence (SIPS)--influence of SIPS on radiotherapy.

PubMed

Suzuki, Masatoshi; Boothman, David A

2008-03-01

Replicative senescence is a fundamental feature in normal human diploid cells and results from dysfunctional telomeres at the Hayflick cell division limit. Ionizing radiation (IR) prematurely induces the same phenotypes as replicative senescence prior to the Hayflick limit. This process is known as stress-induced premature senescence (SIPS). Since the cell cycle is irreversibly arrested in SIPS-induced cells, even if they are stimulated by various growth factors, it is thought that SIPS is a form of cell death, irreversibly eliminating replicating cells. IR-induced-focus formation of DNA repair proteins, a marker of DNA damage, is detected in SIPS as well as replicative senescent cells. Furthermore, both processes persistently induce cell cycle checkpoint mechanisms, indicating DNA damage created by ionizing radiation induces SIPS in normal cells, possibly by the same mechanisms as those occurring in replicative senescence. Interestingly, IR induces SIPS not only in normal cells, but also in tumor cells. Due to the expression of telomerase in tumor cells, telomere-dependent replicative senescence does not occur. However, SIPS is induced under certain conditions after IR exposure. Thus, cell death triggered by IR can be attributed to apoptosis or SIPS in tumor cells. However, metabolic function remains intact in SIPS-induced cancer cells, and recent studies show that senescence eliminate cells undergoing SIPS secrete various kinds of factors outside the cell, changing the microenvironment. Evidence using co-culture systems containing normal senescent stromal cells and epithelial tumor cells show that factors secreted from senescent stroma cells promote the growth of tumor epithelial cells both in vitro and in vivo. Thus, regulation of factors secreted from SIPS-induced stromal cells, as well as tumor cells, may affect radiotherapy.

A synthetic chalcone, 2'-hydroxy-2,3,5'-trimethoxychalcone triggers unfolded protein response-mediated apoptosis in breast cancer cells.

PubMed

Lee, Da Hyun; Jung Jung, You; Koh, Dongsoo; Lim, Yoongho; Lee, Young Han; Shin, Soon Young

2016-03-01

The primary aim of this study was to find novel chemopreventive agents effective against breast cancer. Endoplasmic reticulum (ER) stress can induce apoptosis through the unfolded protein response (UPR). 2'-Hydroxy-2,3,5'-trimethoxychalcone (DK143) is a synthetic flavonoid derivative. The present study provides evidence supporting the role of the UPR in mediating the apoptotic effect of DK143. Treatment with DK143 triggered apoptosis through the activation of the caspase pathway in MDA-MB-231 breast cancer cells without affecting viability of MCF10A non-transformed breast epithelial cells. Further analysis revealed that DK143 produced reactive oxygen species (ROS) in MDA-MB-231 cells, but not in MCF10A cells, and upregulated the expression of ER stress sensors, including GRP78/BiP, IRE1α, CHOP, and Bim in MDA-MB-231 cells. In addition, UPR-related transcription factors, XBP-1 and CHOP, were activated by DK143. Moreover, silencing of IRE1α or CHOP by corresponding siRNA molecules attenuated DK143-induced apoptosis. Furthermore, DK143 suppressed mouse tumor growth in vivo. These results demonstrate that promoting ER stress in breast cancer cells via UPR induction might be a promising strategy for developing new chemotherapeutic or chemopreventive agents for breast cancer. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

IFNβ/TNFα synergism induces a non-canonical STAT2/IRF9-dependent pathway triggering a novel DUOX2 NADPH Oxidase-mediated airway antiviral response

PubMed Central

Fink, Karin; Martin, Lydie; Mukawera, Esperance; Chartier, Stéfany; De Deken, Xavier; Brochiero, Emmanuelle; Miot, Françoise; Grandvaux, Nathalie

2013-01-01

Airway epithelial cells are key initial innate immune responders in the fight against respiratory viruses, primarily via the secretion of antiviral and proinflammatory cytokines that act in an autocrine/paracrine fashion to trigger the establishment of an antiviral state. It is currently thought that the early antiviral state in airway epithelial cells primarily relies on IFNβ secretion and the subsequent activation of the interferon-stimulated gene factor 3 (ISGF3) transcription factor complex, composed of STAT1, STAT2 and IRF9, which regulates the expression of a panoply of interferon-stimulated genes encoding proteins with antiviral activities. However, the specific pathways engaged by the synergistic action of different cytokines during viral infections, and the resulting physiological outcomes are still ill-defined. Here, we unveil a novel delayed antiviral response in the airways, which is initiated by the synergistic autocrine/paracrine action of IFNβ and TNFα, and signals through a non-canonical STAT2- and IRF9-dependent, but STAT1-independent cascade. This pathway ultimately leads to the late induction of the DUOX2 NADPH oxidase expression. Importantly, our study uncovers that the development of the antiviral state relies on DUOX2-dependent H2O2 production. Key antiviral pathways are often targeted by evasion strategies evolved by various pathogenic viruses. In this regard, the importance of the novel DUOX2-dependent antiviral pathway is further underlined by the observation that the human respiratory syncytial virus is able to subvert DUOX2 induction. PMID:23545780

Differential cellular responses by oncogenic levels of c-Myc expression in long-term confluent retinal pigment epithelial cells.

PubMed

Wang, Yiping; Cheng, Xiangdong; Samma, Muhammad Kaleem; Kung, Sam K P; Lee, Clement M; Chiu, Sung Kay

2018-06-01

c-Myc is a highly pleiotropic transcription factor known to control cell cycle progression, apoptosis, and cellular transformation. Normally, ectopic expression of c-Myc is associated with promoting cell proliferation or triggering cell death via activating p53. However, it is not clear how the levels of c-Myc lead to different cellular responses. Here, we generated a series of stable RPE cell clones expressing c-Myc at different levels, and found that consistent low level of c-Myc induced cellular senescence by activating AP4 in post-confluent RPE cells, while the cells underwent cell death at high level of c-Myc. In addition, high level of c-Myc could override the effect of AP4 on cellular senescence. Further knockdown of AP4 abrogated senescence-like phenotype in cells expressing low level of c-Myc, and accelerated cell death in cells with medium level of c-Myc, indicating that AP4 was required for cellular senescence induced by low level of c-Myc.

Simulation of Cell Patterning Triggered by Cell Death and Differential Adhesion in Drosophila Wing.

PubMed

Nagai, Tatsuzo; Honda, Hisao; Takemura, Masahiko

2018-02-27

The Drosophila wing exhibits a well-ordered cell pattern, especially along the posterior margin, where hair cells are arranged in a zigzag pattern in the lateral view. Based on an experimental result observed during metamorphosis of Drosophila, we considered that a pattern of initial cells autonomously develops to the zigzag pattern through cell differentiation, intercellular communication, and cell death (apoptosis) and performed computer simulations of a cell-based model of vertex dynamics for tissues. The model describes the epithelial tissue as a monolayer cell sheet of polyhedral cells. Their vertices move according to equations of motion, minimizing the sum total of the interfacial and elastic energies of cells. The interfacial energy densities between cells are introduced consistently with an ideal zigzag cell pattern, extracted from the experimental result. The apoptosis of cells is modeled by gradually reducing their equilibrium volume to zero and by assuming that the hair cells prohibit neighboring cells from undergoing apoptosis. Based on experimental observations, we also assumed wing elongation along the proximal-distal axis. Starting with an initial cell pattern similar to the micrograph experimentally obtained just before apoptosis, we carried out the simulations according to the model mentioned above and successfully reproduced the ideal zigzag cell pattern. This elucidates a physical mechanism of patterning triggered by cell apoptosis theoretically and exemplifies, to our knowledge, a new framework to study apoptosis-induced patterning. We conclude that the zigzag cell pattern is formed by an autonomous communicative process among the participant cells. Copyright © 2018 Biophysical Society. All rights reserved.

Effects of white light-emitting diode (LED) light exposure with different correlated color temperatures (CCTs) on human lens epithelial cells in culture.

PubMed

Xie, Chen; Li, Xiuyi; Tong, Jianping; Gu, Yangshun; Shen, Ye

2014-01-01

Cataract is the major cause for legal blindness in the world. Oxidative stress on the lens epithelial cells (hLECs) is the most important factor in cataract formation. Cumulative light-exposure from widely used light-emitting diodes (LEDs) may pose a potential oxidative threat to the lens epithelium, due to the high-energy blue light component in the white-light emission from diodes. In the interest of perfecting biosafety standards for LED domestic lighting, this study analyzed the photobiological effect of white LED light with different correlated color temperatures (CCTs) on cultured hLECs. The hLECs were cultured and cumulatively exposed to multichromatic white LED light with CCTs of 2954, 5624, and 7378 K. Cell viability of hLECs was measured by Cell Counting Kit-8 (CCK-8) assay. DNA damage was determined by alkaline comet assay. Intracellular reactive oxygen species (ROS) generation, cell cycle, and apoptosis were quantified by flow cytometry. Compared with 2954 and 5624 K LED light, LED light having a CCT of 7378 K caused overproduction of intracellular ROS and severe DNA damage, which triggered G2 /M arrest and apoptosis. These results indicate that white LEDs with a high CCT could cause significant photobiological damage to hLECs. © 2014 The American Society of Photobiology.

RIP3 AND pMLKL promote necroptosis-induced inflammation and alter membrane permeability in intestinal epithelial cells.

PubMed

Negroni, Anna; Colantoni, Eleonora; Pierdomenico, Maria; Palone, Francesca; Costanzo, Manuela; Oliva, Salvatore; Tiberti, Antonio; Cucchiara, Salvatore; Stronati, Laura

2017-11-01

Necroptosis is an inflammatory form of programmed cell death requiring receptor-interacting protein kinase 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL). The aim of this study is to examine in depth in vitro and ex vivo the contribution of necroptosis to intestinal inflammation. In vitro: we used an intestinal cell line, HCT116RIP3, produced in our laboratory and overexpressing RIP3. Ex vivo: intestinal mucosal biopsies were taken from patients with inflammatory bowel disease (IBD) (20 with Crohn's disease; 20 with ulcerative colitis) and from 20 controls. RIP3-induced necroptosis triggers MLKL activation, increases cytokine/alarmin expression (IL-8, IL-1β, IL-33, HMGB1), NF-kBp65 translocation and NALP3 inflammasome assembly. It also affects membrane permeability by altering cell-cell junctional proteins (E-cadherin, Occludin, Zonulin-1). Targeting necroptosis through Necrostatin-1 significantly reduces intestinal inflammation in vitro and in cultured intestinal explants from IBD. We show for the first time in vitro and ex vivo that RIP3-driven necroptosis seriously affects intestinal inflammation by increasing pMLKL, activating different cytokines and alarmins, and altering epithelial permeability. The inhibition of necroptosis causes a significant decrease of all these effects. These data strongly support the view that targeting necroptosis may represent a promising new option for the treatment of inflammatory enteropathies. Copyright © 2017. Published by Elsevier Ltd.

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

PubMed

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

2016-05-06

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

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

PubMed

Ito, Takuji; Bai, Tao; Tanaka, Tetsuji; Yoshida, Kenji; Ueyama, Takashi; Miyajima, Masayasu; Negishi, Takayuki; Kawasaki, Takahiko; Takamatsu, Hyota; Kikutani, Hitoshi; Kumanogoh, Atsushi; Yukawa, Kazunori

2015-02-01

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

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

PubMed Central

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

2015-01-01

The opening of the mouse vaginal cavity to the skin is a postnatal tissue remodeling process that occurs at approximately five weeks of age for the completion of female genital tract maturation at puberty. The tissue remodeling process is primarily composed of a hormonally triggered apoptotic process predominantly occurring in the epithelium of the distal section of the vaginal cavity. However, the detailed mechanism underlying the apoptotic induction remains to be elucidated. In the present study, it was observed that the majority of BALB/c mice lacking the class 4 semaphorin, semaphorin 4D (Sema4D), developed imperforate vagina and hydrometrocolpos resulting in a perpetually unopened vaginal cavity regardless of a normal estrogen level comparable with that in wild-type (WT) mice. Administration of β-estradiol to infant Sema4D-deficient (Sema4D−/−) mice did not induce precocious vaginal opening, which was observed in WT mice subjected to the same β-estradiol administration, excluding the possibility that the closed vaginal phenotype was due to insufficient estrogen secretion at the time of vaginal opening. In order to assess the role of Sema4D in the postnatal vaginal tissue remodeling process, the expression of Sema4D and its receptor, plexin-B1, was examined as well as the level of apoptosis in the vaginal epithelia of five-week-old WT and Sema4D−/− mice. Immunohistochemical analyses confirmed the localization of Sema4D and plexin-B1 in the mouse vaginal epithelia. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunohistochemistry detecting activated caspase-3 revealed significantly fewer apoptotic cells in situ in the vaginal mucosa of five-week-old Sema4D−/− mice compared with WT mice. The addition of recombinant Sema4D to Sema4D−/− vaginal epithelial cells in culture significantly enhanced apoptosis of the vaginal epithelial cells, demonstrating the apoptosis-inducing activity of Sema4D. The experimental reduction of plexin-B1 expression in vaginal epithelial cells demonstrated the integral role of plexin-B1 in Sema4D-induced apoptotic cell death. These results suggest a non-redundant role of Sema4D in the postnatal tissue remodeling process in five-week-old BALB/c mice, which involves the induction of vaginal epithelial cell apoptosis through Sema4D binding to plexin-B1. PMID:25351707

Leukemia inhibitory factor: part of a large ingathering family.

PubMed

Taupin, J L; Pitard, V; Dechanet, J; Miossec, V; Gualde, N; Moreau, J F

1998-01-01

Leukemia Inhibitory Factor (LIF) has a wide variety of biological activities. It regulates the differentiation of embryonic stem cells, neural cells, osteoblasts, adipocytes, hepatocytes and kidney epithelial cells. It also triggers the proliferation of myoblasts, primordial germ cells and some endothelial cells. Many of these biological functions parallel those of interleukin-6, Oncostatin M, ciliary neurotrophic factor, interleukin-11 and cardiotrophin-1. These structurally related cytokines also share subunits of their receptors which could partially explain the redundancy in this system of soluble mediators. In vivo LIF proves important in regulating the inflammatory response by fine tuning of the delicate balance of at least four systems in the body, namely the immune, the hematopoietic, the nervous and the endocrine systems. Although we are far from its therapeutic applications, the fast increasing knowledge in this field may bring new insights for the understanding of the cytokine biology in general.

From single cells to tissue architecture-a bottom-up approach to modelling the spatio-temporal organisation of complex multi-cellular systems.

PubMed

Galle, J; Hoffmann, M; Aust, G

2009-01-01

Collective phenomena in multi-cellular assemblies can be approached on different levels of complexity. Here, we discuss a number of mathematical models which consider the dynamics of each individual cell, so-called agent-based or individual-based models (IBMs). As a special feature, these models allow to account for intracellular decision processes which are triggered by biomechanical cell-cell or cell-matrix interactions. We discuss their impact on the growth and homeostasis of multi-cellular systems as simulated by lattice-free models. Our results demonstrate that cell polarisation subsequent to cell-cell contact formation can be a source of stability in epithelial monolayers. Stroma contact-dependent regulation of tumour cell proliferation and migration is shown to result in invasion dynamics in accordance with the migrating cancer stem cell hypothesis. However, we demonstrate that different regulation mechanisms can equally well comply with present experimental results. Thus, we suggest a panel of experimental studies for the in-depth validation of the model assumptions.

Lacritin and other new proteins of the lacrimal functional unit.

PubMed

McKown, Robert L; Wang, Ningning; Raab, Ronald W; Karnati, Roy; Zhang, Yinghui; Williams, Patricia B; Laurie, Gordon W

2009-05-01

The lacrimal functional unit (LFU) is defined by the 2007 International Dry Eye WorkShop as 'an integrated system comprising the lacrimal glands, ocular surface (cornea, conjunctiva and meibomian glands) and lids, and the sensory and motor nerves that connect them'. The LFU maintains a healthy ocular surface primarily through a properly functioning tear film that provides protection, lubrication, and an environment for corneal epithelial cell renewal. LFU cells express thousands of proteins. Over 200 new LFU proteins have been discovered in the last decade. Lacritin is a new LFU-specific growth factor in human tears that flows through ducts to target corneal epithelial cells on the ocular surface. When applied topically in rabbits, lacritin appears to increase the volume of basal tear secretion. Lacritin is one of only a handful of tear proteins preliminarily reported to be downregulated in blepharitis and in two dry eye syndromes. Computational analysis predicts an ordered C-terminal domain that binds the corneal epithelial cell surface proteoglycan syndecan-1 (SDC1) and is required for lacritin's low nanomolar mitogenic activity. The lacritin-binding site on the N-terminus of SDC1 is exposed by heparanase. Heparanase is constitutively expressed by the corneal epithelium and appears to be a normal constituent of tears. Binding triggers rapid signaling to downstream NFAT and mTOR. A wealth of other new proteins, originally designated as hypothetical when first identified by genomic sequencing, are expressed by the human LFU including: ALS2CL, ARHGEF19, KIAA1109, PLXNA1, POLG, WIPI1 and ZMIZ2. Their demonstrated or implied roles in human genetic disease or basic cellular functions are fuel for new investigation. Addressing topical areas in ocular surface physiology with new LFU proteins may reveal interesting new biological mechanisms and help get to the heart of ocular surface dysfunction.

Pigment Epithelial-derived Factor (PEDF)-triggered Lung Cancer Cell Apoptosis Relies on p53 Protein-driven Fas Ligand (Fas-L) Up-regulation and Fas Protein Cell Surface Translocation*

PubMed Central

Li, Lei; Yao, Ya-Chao; Fang, Shu-Huan; Ma, Cai-Qi; Cen, Yi; Xu, Zu-Min; Dai, Zhi-Yu; Li, Cen; Li, Shuai; Zhang, Ting; Hong, Hong-Hai; Qi, Wei-Wei; Zhou, Ti; Li, Chao-Yang; Yang, Xia; Gao, Guo-Quan

2014-01-01

Pigment epithelium-derived factor (PEDF), a potent antiangiogenesis agent, has recently attracted attention for targeting tumor cells in several types of tumors. However, less is known about the apoptosis-inducing effect of PEDF on human lung cancer cells and the underlying molecular events. Here we report that PEDF has a growth-suppressive and proapoptotic effect on lung cancer xenografts. Accordingly, in vitro, PEDF apparently induced apoptosis in A549 and Calu-3 cells, predominantly via the Fas-L/Fas death signaling pathway. Interestingly, A549 and Calu-3 cells are insensitive to the Fas-L/Fas apoptosis pathway because of the low level of cell surface Fas. Our results revealed that, in addition to the enhancement of Fas-L expression, PEDF increased the sensitivity of A549 and Calu-3 cells to Fas-L-mediated apoptosis by triggering the translocation of Fas protein to the plasma membrane in a p53- and FAP-1-dependent manner. Similarly, the up-regulation of Fas-L by PEDF was also mediated by p53. Furthermore, peroxisome proliferator-activated receptor γ was determined to be the upstream regulator of p53. Together, these findings uncover a novel mechanism of tumor cell apoptosis induced by PEDF and provide a potential therapeutic strategy for tumors that are insensitive to Fas-L/Fas-dependent apoptosis because of a low level of cell surface Fas. PMID:25225287

Radon-induced reduced apoptosis in human bronchial epithelial cells with knock-down of mitochondria DNA

PubMed Central

Li, Bing-Yan; Sun, Jing; Wei, Hong; Cheng, Yu-Zhi; Xue, Lian; Cheng, Zhi-Hai; Wan, Jian-Mei; Wang, Ai-Qing; Hei, Tom K.; Tong, Jian

2012-01-01

Radon and radon progeny inhalation exposure are recognized to induce lung cancer. To explore the role of mitochondria in radon-induced carcinogenesis in humans, an in vitro partially depleted mitochondrial DNA (mtDNA) cell line (ρ−) was generated by treatment of human bronchial epithelial (HBE) cells (ρ+) with ethidium bromide (EB). The characterization of ρ− cells indicated the presence of dysfunctional mitochondria and might thus serve a reliable model to investigate the role of mitochondria. In a gas inhalation chamber, ρ− and ρ+ cells were exposed to radon gas produced by a radium source. Results showed that apoptosis was significantly increased both in ρ− and ρ+ cells irradiated by radon. Moreover, apoptosis in ρ− cells showed a lower level than in ρ+ cells. Radon was further found to depress mitochondrial membrane potential (MMP) of HBE cells with knock-down mtDNA. Production of reactive oxygen species (ROS) was markedly elevated both in ρ− and ρ+ cells exposed to radon. The distribution of phases of cell cycle was different in ρ− compared to ρ+ cells. Radon-irradiation induced a rise in G2/M and decrease in S phase in ρ+ cells. In ρ− cells, G1, G2/M and S populations remained similar to cells exposed to radon. In conclusion, radon-induced changes in ROS generation, MMP and cell cycle are all attributed to reduction of apoptosis which may trigger and promote cell transformation leading to carcinogenesis. Our study indicates that the use of the ρ− knock-down mtDNA HBE cells may serve as a reliable model to study the role played by mitochondria in carcinogenic diseases. PMID:22891884

Cellular and Molecular effects of Vitamin D on Carcinogenesis

PubMed Central

Welsh, JoEllen

2011-01-01

Epidemiologic data suggest that the incidence and severity of many types of cancer inversely correlates with indices of vitamin D status. The vitamin D receptor (VDR) is highly expressed in epithelial cells at risk for carcinogenesis including those resident in skin, breast, prostate and colon, providing a direct molecular link by which vitamin D status impacts on carcinogenesis. Consistent with this concept, activation of VDR by its ligand 1,25-dihydroxyvitamin D (1,25D) triggers comprehensive genomic changes in epithelial cells that contribute to maintenance of the differentiated phenotype, resistance to cellular stresses and protection of the genome. Many epithelial cells also express the vitamin D metabolizing enzyme CYP27B1 which enables autocrine generation of 1,25D from the circulating vitamin D metabolite 25-hydroxyvitamin D (25D), critically linking overall vitamin D status with cellular anti-tumor actions. Furthermore, pre-clinical studies in animal models has demonstrated that dietary supplementation with vitamin D or chronic treatment with VDR agonists decreases tumor development in skin, colon, prostate and breast. Conversely, deletion of the VDR gene in mice alters the balance between proliferation and apoptosis, increases oxidative DNA damage, and enhances susceptibility to carcinogenesis in these tissues. Because VDR expression is retained in many human tumors, vitamin D status may be an important modulator of cancer progression in persons living with cancer. Collectively, these observations have reinforced the need to further define the molecular actions of the VDR and the human requirement for vitamin D in relation to cancer development and progression. PMID:22085499

Hypoxia attenuates inflammatory mediators production induced by Acanthamoeba via Toll-like receptor 4 signaling in human corneal epithelial cells

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

Pan, Hong; The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Public Health, Qilu Hospital, Shandong University, 107, Wenhua Xi Road, Jinan 250012; Wu, Xinyi, E-mail: xywu8868@163.com

2012-04-13

Highlights: Black-Right-Pointing-Pointer Hypoxia attenuates Acanthamoeba-induced the production of IL-8 and IFN-{beta}. Black-Right-Pointing-Pointer Hypoxia inhibits TLR4 expression in a time-dependent manner in HCECs. Black-Right-Pointing-Pointer Hypoxia inhibits Acanthamoeba-induced the activation of NF-{kappa}B and ERK1/2 in HCECs. Black-Right-Pointing-Pointer Hypoxia decreases Acanthamoeba-induced inflammatory response via TLR4 signaling. Black-Right-Pointing-Pointer LPS-induced the secretion of IL-6 and IL-8 is abated by hypoxia via TLR4 signaling. -- Abstract: Acanthamoeba keratitis (AK) is a vision-threatening corneal infection that is intimately associated with contact lens use which leads to hypoxic conditions on the corneal surface. However, the effect of hypoxia on the Acanthamoeba-induced host inflammatory response of corneal epithelial cellsmore » has not been studied. In the present study, we investigated the effect of hypoxia on the Acanthamoeba-induced production of inflammatory mediators interleukin-8 (IL-8) and interferon-{beta} (IFN-{beta}) in human corneal epithelial cells and then evaluated its effects on the Toll-like receptor 4 (TLR4) signaling, including TLR4 and myeloid differentiation primary response gene (88) (MyD88) expression as well as the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-{kappa}B) and extracellular signal-regulated kinases 1/2 (ERK1/2). We then studied the effect of hypoxia on a TLR4-specific inflammatory response triggered by the TLR4 ligand lipopolysaccharide (LPS). Our data showed that hypoxia significantly decreased the production of IL-8 and IFN-{beta}. Furthermore, hypoxia attenuated Acanthamoeba-triggered TLR4 expression as well as the activation of NF-{kappa}B and ERK1/2, indicating that hypoxia abated Acanthamoeba-induced inflammatory responses by affecting TLR4 signaling. Hypoxia also inhibited LPS-induced IL-6 and IL-8 secretion, myeloid differentiation primary response gene (88) MyD88 expression and NF-{kappa}B activation, confirming that hypoxia suppressed the LPS-induced inflammatory response by affecting TLR4 signaling. In conclusion, our results demonstrated that hypoxia attenuated the host immune and inflammatory response against Acanthamoeba infection by suppressing TLR4 signaling, indicating that hypoxia might impair the host cell's ability to eliminate the Acanthamoeba invasion and that hypoxia could enhance cell susceptibility to Acanthamoeba infection. These results may explain why contact lens use is one of the most prominent risk factors for AK.« less

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

PubMed

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

2016-07-31

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

Interaction with Epithelial Cells Modifies Airway Macrophage Response to Ozone

EPA Science Inventory

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

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

PubMed

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

2010-07-02

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

Re-establishment of gap junctional intercellular communication (GJIC) between human endometrial carcinomas by prostaglandin E(2).

PubMed

Schlemmer, Scott R; Kaufman, David G

2012-12-01

Reduced intercellular communication via gap junctions is correlated with carcinogenesis. Gap junctional intercellular communication (GJIC), between normal human endometrial epithelial cells is enhanced when endometrial stromal cells were present in culture. This enhancement of GJIC between normal epithelial cells also occurs when they are cultured in medium conditioned by stromal cells. This observation indicated that a soluble compound (or compounds) produced and secreted by stromal cells mediates GJIC in epithelial cells. Previous studies have shown that endometrial stromal cells release prostaglandin E(2) (PGE(2)) and prostaglandin F(2α) (PGF(2α)) under physiological conditions. When we evaluated the response of normal endometrial epithelial cells to various concentrations of PGE(2,) we found enhanced GJIC with 1nM PGE(2). This is a smaller increase in GJIC than that induced by medium conditioned by stromal cells. When the extracellular concentration of PGE(2) was measured after incubation with stromal cells, it was found to be similar to the concentrations showing maximal GJIC between the normal epithelial cells. When indomethacin was used to inhibit prostaglandin synthesis by stromal cells, GJIC was reduced but not eliminated between normal endometrial epithelial cells. These observations suggest that although PGE(2) secreted by stromal cells is an important mediator of GJIC between the epithelial cells, it is not the sole mediator. Transformed endometrial epithelial cells did not demonstrate GJIC even in the presence of stromal cells. However, we were able to re-establish GJIC in transformed epithelial cells when we added PGE(2) to the cells. Our findings show that PGE(2) may serve as an intercellular mediator between stromal and epithelial cells that regulates GJIC in normal and malignant epithelial cells. This suggests that maintenance of GJIC by preserving or replacing PGE(2) secretion by endometrial stromal cells may have the potential to suppress carcinogenesis in endometrial epithelial cells. Copyright © 2012 Elsevier Inc. All rights reserved.

Polyamine-dependent migration of retinal pigment epithelial cells.

PubMed

Johnson, Dianna A; Fields, Carolyn; Fallon, Amy; Fitzgerald, Malinda E C; Viar, Mary Jane; Johnson, Leonard R

2002-04-01

Migration of retinal pigment epithelial (RPE) cells can be triggered by disruption of the RPE monolayer or injury to the neural retina. Migrating cells may re-establish a confluent monolayer, or they may invade the neural retina and disrupt visual function. The purpose of this study was to examine the role of endogenous polyamines in mechanisms of RPE migration. Endogenous polyamine levels were determined in an immortalized RPE cell line, D407, using HPLC. Activities of the two rate-limiting enzymes for polyamine synthesis, ornithine decarboxylase (ODC), and S-adenosylmethionine decarboxylase (SAMdc), were measured by liberation of ((14)CO(2))(.) Migration was assessed in confluent cultures by determining the number of cells migrating into a mechanically denuded area. All measurements were obtained both in control cultures and in cultures treated with synthesis inhibitors that deplete endogenous polyamines. Subcellular localization of endogenous polyamines was determined using a polyamine antibody. The polyamines, spermidine and spermine, as well as their precursor, putrescine, were normal constituents of RPE cells. The two rate-limiting synthetic enzymes were also present, and their activities were stimulated dramatically by addition of serum to the culture medium. Cell migration was similarly stimulated by serum exposure. When endogenous polyamines were depleted, migration was blocked. When polyamines were replenished through uptake, migration was restored. Polyamine immunoreactivity was limited to membrane patches in quiescent cells. In actively migrating and dividing cells, immunoreactivity was enhanced throughout the cytoplasm. Polyamines are essential for RPE migration. Pharmacologic manipulation of the polyamine pathway could provide a therapeutic strategy for regulating anomalous migration.

Mechanical stress as a regulator of cell motility

NASA Astrophysics Data System (ADS)

Putelat, T.; Recho, P.; Truskinovsky, L.

2018-01-01

The motility of a cell can be triggered or inhibited not only by an applied force but also by a mechanically neutral force couple. This type of loading, represented by an applied stress and commonly interpreted as either squeezing or stretching, can originate from extrinsic interaction of a cell with its neighbors. To quantify the effect of applied stresses on cell motility we use an analytically transparent one-dimensional model accounting for active myosin contraction and induced actin turnover. We show that stretching can polarize static cells and initiate cell motility while squeezing can symmetrize and arrest moving cells. We show further that sufficiently strong squeezing can lead to the loss of cell integrity. The overall behavior of the system depends on the two dimensionless parameters characterizing internal driving (chemical activity) and external loading (applied stress). We construct a phase diagram in this parameter space distinguishing between static, motile, and collapsed states. The obtained results are relevant for the mechanical understanding of contact inhibition and the epithelial-to-mesenchymal transition.

Hedgehog-mediated mesenchymal-epithelial interactions modulate hepatic response to bile duct ligation.

PubMed

Omenetti, Alessia; Yang, Liu; Li, Yin-Xiong; McCall, Shannon J; Jung, Youngmi; Sicklick, Jason K; Huang, Jiawen; Choi, Steve; Suzuki, Ayako; Diehl, Anna Mae

2007-05-01

In bile duct-ligated (BDL) rodents, as in humans with chronic cholangiopathies, biliary obstruction triggers proliferation of bile ductular cells that are surrounded by fibrosis produced by adjacent myofibroblastic cells in the hepatic mesenchyme. The proximity of the myofibroblasts and cholangiocytes suggests that mesenchymal-epithelial crosstalk promotes the fibroproliferative response to cholestatic liver injury. Studying BDL mice, we found that bile duct obstruction induces activity of the Hedgehog (Hh) pathway, a system that regulates the viability and differentiation of various progenitors during embryogenesis. After BDL, many bile ductular cells and fibroblastic-appearing cells in the portal stroma express Hh ligands, receptor and/or target genes. Transwell cocultures of an immature cholangiocyte line that expresses the Hh receptor, Patched (Ptc), with liver myofibroblastic cells demonstrated that both cell types produced Hh ligands that enhanced each other's viability and proliferation. Further support for the concept that Hh signaling modulates the response to BDL was generated by studying PtcLacZ mice, which have an impaired ability to constrain Hh signaling due to a heterozygous deficiency of Ptc. After BDL, PtcLacZ mice upregulated fibrosis gene expression earlier than wild-type controls and manifested an unusually intense ductular reaction, more expanded fibrotic portal areas, and a greater number of lobular necrotic foci. Our findings reveal that adult livers resurrect developmental signaling systems, such as the Hh pathway, to guide remodeling of the biliary epithelia and stroma after cholestatic injury.

The PI3K Pathway Balances Self-Renewal and Differentiation of Nephron Progenitor Cells through β-Catenin Signaling

PubMed Central

Lindström, Nils Olof; Carragher, Neil Oliver; Hohenstein, Peter

2015-01-01

Summary Nephron progenitor cells differentiate to form nephrons during embryonic kidney development. In contrast, self-renewal maintains progenitor numbers and premature depletion leads to impaired kidney function. Here we analyze the PI3K pathway as a point of convergence for the multiple pathways that are known to control self-renewal in the kidney. We demonstrate that a reduction in PI3K signaling triggers premature differentiation of the progenitors and activates a differentiation program that precedes the mesenchymal-to-epithelial transition through ectopic activation of the β-catenin pathway. Therefore, the combined output of PI3K and other pathways fine-tunes the balance between self-renewal and differentiation in nephron progenitors. PMID:25754203

Lactobacillus rhamnosus GG protects the intestinal epithelium from radiation injury through release of lipoteichoic acid, macrophage activation and the migration of mesenchymal stem cells.

PubMed

Riehl, Terrence E; Alvarado, David; Ee, Xueping; Zuckerman, Aaron; Foster, Lynn; Kapoor, Vaishali; Thotala, Dinesh; Ciorba, Matthew A; Stenson, William F

2018-06-22

Lactobacillus rhamnosus GG (LGG), a probiotic, given by gavage is radioprotective of the mouse intestine. LGG-induced radioprotection is toll-like receptor 2 (TLR2) and cyclooxygenase-2 (COX-2)-dependent and is associated with the migration of COX-2+mesenchymal stem cells (MSCs) from the lamina propria of the villus to the lamina propria near the crypt epithelial stem cells. Our goals were to define the mechanism of LGG radioprotection including identification of the TLR2 agonist, and the mechanism of the MSC migration and to determine the safety and efficacy of this approach in models relevant to clinical radiation therapy. Intestinal radioprotection was modelled in vitro with cell lines and enteroids as well as in vivo by assaying clinical outcomes and crypt survival. Fractionated abdominal and single dose radiation were used along with syngeneic CT26 colon tumour grafts to assess tumour radioprotection. LGG with a mutation in the processing of lipoteichoic acid (LTA), a TLR2 agonist, was not radioprotective, while LTA agonist and native LGG were. An agonist of CXCR4 blocked LGG-induced MSC migration and LGG-induced radioprotection. LGG given by gavage induced expression of CXCL12, a CXCR4 agonist, in pericryptal macrophages and depletion of macrophages by clodronate liposomes blocked LGG-induced MSC migration and radioprotection. LTA effectively protected the normal intestinal crypt, but not tumours in fractionated radiation regimens. LGG acts as a 'time-release capsule' releasing radioprotective LTA. LTA then primes the epithelial stem cell niche to protect epithelial stem cells by triggering a multicellular, adaptive immune signalling cascade involving macrophages and PGE2 secreting MSCs. NCT01790035; Pre-results. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Taurine protects HK-2 cells from oxidized LDL-induced cytotoxicity via the ROS-mediated mitochondrial and p53-related apoptotic pathways

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

Chang, Chun-Yu; Shen, Chao-Yu; Department of Medical Imaging, Chung Shan Medical University Hospital, Taichung, Taiwan

Oxidized LDL (oxLDL) induces a pro-oxidative environment and promotes apoptosis, causing the progression of renal diseases in humans. Taurine is a semi-essential amino acid in mammals and has been shown to be a potent endogenous antioxidant. The kidney plays a pivotal role in maintaining the balance of taurine. However, the mechanisms underlying the protective effects of taurine against oxLDL-induced injury in renal epithelial cells have not been clarified. In the present study, we investigated the anti-apoptotic effects of taurine on human proximal tubular epithelial (HK-2) cells exposed to oxLDL and explored the related mechanisms. We observed that oxLDL increased themore » contents of ROS and of malondialdehyde (MDA), which is a lipid peroxidation by-product that acts as an indicator of the cellular oxidation status. In addition, oxLDL induced cell death and apoptosis in HK-2 cells. Pretreatment with taurine at 100 μM significantly attenuated the oxLDL-induced cytotoxicity. We determined that oxLDL triggered the phosphorylation of ERK and, in turn, the activation of p53 and other apoptosis-related events, including calcium accumulation, destabilization of the mitochondrial permeability and disruption of the balance between pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins. The malfunctions induced by oxLDL were effectively blocked by taurine. Thus, our results suggested that taurine exhibits potential therapeutic activity by preventing oxLDL-induced nephrotoxicity. The inhibition of oxLDL-induced epithelial apoptosis by taurine was at least partially due to its anti-oxidant activity and its ability to modulate the ERK and p53 apoptotic pathways. - Highlights: • Oxidized LDL induced cytotoxicity and apoptosis in HK-2 cells. • Pretreatment with taurine attenuated oxLDL-induced nephrotoxicity. • Taurine protected against renal damages through inhibition of ROS generation. • Taurine prevented apoptosis through modulation of the p53 phosphorylation.« less

Targeting palmitoyl acyltransferase ZDHHC21 improves gut epithelial barrier dysfunction resulting from burn-induced systemic inflammation.

PubMed

Haines, R J; Wang, C Y; Yang, C G Y; Eitnier, R A; Wang, F; Wu, M H

2017-12-01

Clinical studies in burn patients demonstrate a close association between leaky guts and increased incidence or severity of sepsis and other complications. Severe thermal injury triggers intestinal inflammation that contributes to intestinal epithelial hyperpermeability, which exacerbates systemic response leading to multiple organ failure and sepsis. In this study, we identified a significant function of a particular palmitoyl acyltransferase, zinc finger DHHC domain-containing protein-21 (ZDHHC21), in mediating signaling events required for gut hyperpermeability induced by inflammation. Using quantitative PCR, we show that ZDHHC21 mRNA production was enhanced twofold when intestinal epithelial cells were treated with TNF-α-IFN-γ in vitro. In addition, pharmacological targeting of palmitoyl acyltransferases with 2-bromopalmitate (2-BP) showed significant improvement in TNF-α-IFN-γ-mediated epithelial barrier dysfunction by using electric cell-substrate impedance-sensing assays, as well as FITC-labeled dextran permeability assays. Using acyl-biotin exchange assay and click chemistry, we show that TNF-α-IFN-γ treatment of intestinal epithelial cells results in enhanced detection of total palmitoylated proteins and this response is inhibited by 2-BP. Using ZDHHC21-deficient mice or wild-type mice treated with 2-BP, we showed that mice with impaired ZDHHC21 expression or pharmacological inhibition resulted in attenuated intestinal barrier dysfunction caused by thermal injury. Moreover, hematoxylin and eosin staining of the small intestine, as well as transmission electron microscopy, showed that mice with genetic interruption of ZDHHC21 had attenuated villus structure disorganization associated with thermal injury-induced intestinal barrier damage. Taken together, these results suggest an important role of ZDHHC21 in mediating gut hyperpermeability resulting from thermal injury. NEW & NOTEWORTHY Increased mucosal permeability in the gut is one of the major complications following severe burn. Here we report the novel finding that zinc finger DHHC domain-containing protein-21 (ZDHHC21) mediates gut epithelial hyperpermeability resulting from an experimental model of thermal injury. The hyperpermeability response was significantly attenuated with a pharmacological inhibitor of palmitoyl acyltransferases and in mice with genetic ablation of ZDHHC21. These findings suggest that ZDHHC21 may serve as a novel therapeutic target for treating burn-induced intestinal barrier dysfunction. Copyright © 2017 the American Physiological Society.

Epithelial Cell–Derived Secreted and Transmembrane 1a Signals to Activated Neutrophils during Pneumococcal Pneumonia

PubMed Central

Kamata, Hirofumi; Yamamoto, Kazuko; Wasserman, Gregory A.; Zabinski, Mary C.; Yuen, Constance K.; Lung, Wing Yi; Gower, Adam C.; Belkina, Anna C.; Ramirez, Maria I.; Deng, Jane C.; Quinton, Lee J.; Jones, Matthew R.

2016-01-01

Airway epithelial cell responses are critical to the outcome of lung infection. In this study, we aimed to identify unique contributions of epithelial cells during lung infection. To differentiate genes induced selectively in epithelial cells during pneumonia, we compared genome-wide expression profiles from three sorted cell populations: epithelial cells from uninfected mouse lungs, epithelial cells from mouse lungs with pneumococcal pneumonia, and nonepithelial cells from those same infected lungs. Of 1,166 transcripts that were more abundant in epithelial cells from infected lungs compared with nonepithelial cells from the same lungs or from epithelial cells of uninfected lungs, 32 genes were identified as highly expressed secreted products. Especially strong signals included two related secreted and transmembrane (Sectm) 1 genes, Sectm1a and Sectm1b. Refinement of sorting strategies suggested that both Sectm1 products were induced predominantly in conducting airway epithelial cells. Sectm1 was induced during the early stages of pneumococcal pneumonia, and mutation of NF-κB RelA in epithelial cells did not diminish its expression. Instead, type I IFN signaling was necessary and sufficient for Sectm1 induction in lung epithelial cells, mediated by signal transducer and activator of transcription 1. For target cells, Sectm1a bound to myeloid cells preferentially, in particular Ly6GbrightCD11bbright neutrophils in the infected lung. In contrast, Sectm1a did not bind to neutrophils from uninfected lungs. Sectm1a increased expression of the neutrophil-attracting chemokine CXCL2 by neutrophils from the infected lung. We propose that Sectm1a is an epithelial product that sustains a positive feedback loop amplifying neutrophilic inflammation during pneumococcal pneumonia. PMID:27064756

Graphene-induced apoptosis in lung epithelial cells through EGFR

NASA Astrophysics Data System (ADS)

Tsai, Shih-Ming; Bangalore, Preeti; Chen, Eric Y.; Lu, David; Chiu, Meng-Hsuen; Suh, Andrew; Gehring, Matthew; Cangco, John P.; Garcia, Santiago G.; Chin, Wei-Chun

2017-07-01

Expanding interest in nanotechnology applied to electronic and biomedical fields has led to fast-growing development of various nanomaterials. Graphene is a single-atom thick, two-dimensional sheet of hexagonally arranged carbon atoms with unique physical and chemical properties. Recently, graphene has been used in many studies on electronics, photonics, composite materials, energy generation and storage, sensors, and biomedicine. However, the current health risk assessment for graphene has been relatively limited and inconclusive. This study evaluated the toxicity effects of graphene on the airway epithelial cell line BEAS-2B, which represents the first barrier of the human body to interact with airborne graphene particles. Our result showed that graphene can induce the cellular Ca2+ by phospholipase C (PLC) associated pathway by activating epidermal growth factor receptor (EGFR). Subsequently, inositol 1,4,5-triphosphate (IP3) receptors activate the release of Ca2+ from the endoplasmic reticulum (ER) Ca2+ stores. Those Ca2+ signals further trigger the calcium-regulated apoptosis in the cell. Furthermore, the stimulation can cause EGFR upregulation, which have been demonstrated to associate with diseases such as lung cancer, chronic obstructive pulmonary disease (COPD), and cardiovascular diseases. This study highlights the additional health risk considering that it can function as a contributing factor for other respiratory diseases.

Combined Effects of Ventilation Mode and Positive End-Expiratory Pressure on Mechanics, Gas Exchange and the Epithelium in Mice with Acute Lung Injury

PubMed Central

Thammanomai, Apiradee; Hamakawa, Hiroshi; Bartolák-Suki, Erzsébet; Suki, Béla

2013-01-01

The accepted protocol to ventilate patients with acute lung injury is to use low tidal volume (VT) in combination with recruitment maneuvers or positive end-expiratory pressure (PEEP). However, an important aspect of mechanical ventilation has not been considered: the combined effects of PEEP and ventilation modes on the integrity of the epithelium. Additionally, it is implicitly assumed that the best PEEP-VT combination also protects the epithelium. We aimed to investigate the effects of ventilation mode and PEEP on respiratory mechanics, peak airway pressures and gas exchange as well as on lung surfactant and epithelial cell integrity in mice with acute lung injury. HCl-injured mice were ventilated at PEEPs of 3 and 6 cmH2O with conventional ventilation (CV), CV with intermittent large breaths (CVLB) to promote recruitment, and a new mode, variable ventilation, optimized for mice (VVN). Mechanics and gas exchange were measured during ventilation and surfactant protein (SP)-B, proSP-B and E-cadherin levels were determined from lavage and lung homogenate. PEEP had a significant effect on mechanics, gas exchange and the epithelium. The higher PEEP reduced lung collapse and improved mechanics and gas exchange but it also down regulated surfactant release and production and increased epithelial cell injury. While CVLB was better than CV, VVN outperformed CVLB in recruitment, reduced epithelial injury and, via a dynamic mechanotransduction, it also triggered increased release and production of surfactant. For long-term outcome, selection of optimal PEEP and ventilation mode may be based on balancing lung physiology with epithelial injury. PMID:23326543

Hertwig's Epithelial Root Sheath Fate during Initial Cellular Cementogenesis in Rat Molars.

PubMed

Yamamoto, Tsuneyuki; Yamada, Tamaki; Yamamoto, Tomomaya; Hasegawa, Tomoka; Hongo, Hiromi; Oda, Kimimitsu; Amizuka, Norio

2015-06-29

To elucidate the fate of the epithelial root sheath during initial cellular cementogenesis, we examined developing maxillary first molars of rats by immunohistochemistry for keratin, vimentin, and tissue non-specific alkaline phosphatase (TNALP) and by TdT-mediated dUTP nick end labeling (TUNEL). The advancing root end was divided into three sections, which follow three distinct stages of initial cellular cementogenesis: section 1, where the epithelial sheath is intact; section 2, where the epithelial sheath becomes fragmented; and section 3, where initial cellular cementogenesis begins. After fragmentation of the epithelial sheath, many keratin-positive epithelial sheath cells were embedded in the rapidly growing cellular cementum. A few unembedded epithelial cells located on the cementum surface. Dental follicle cells, precementoblasts, and cementoblasts showed immunoreactivity for vimentin and TNALP. In all three sections, there were virtually no cells possessing double immunoreactivity for vimentin-keratin or TNALP-keratin and only embedded epithelial cells showed TUNEL reactivity. Taken together, these findings suggest that: (1) epithelial sheath cells divide into two groups; one group is embedded in the cementum and thereafter dies by apoptosis, and the other survives on the cementum surface as epithelial cell rests of Malassez; and (2) epithelial sheath cells do not undergo epithelial-mesenchymal transition during initial cellular cementogenesis.

Hertwig’s Epithelial Root Sheath Fate during Initial Cellular Cementogenesis in Rat Molars

PubMed Central

Yamamoto, Tsuneyuki; Yamada, Tamaki; Yamamoto, Tomomaya; Hasegawa, Tomoka; Hongo, Hiromi; Oda, Kimimitsu; Amizuka, Norio

2015-01-01

To elucidate the fate of the epithelial root sheath during initial cellular cementogenesis, we examined developing maxillary first molars of rats by immunohistochemistry for keratin, vimentin, and tissue non-specific alkaline phosphatase (TNALP) and by TdT-mediated dUTP nick end labeling (TUNEL). The advancing root end was divided into three sections, which follow three distinct stages of initial cellular cementogenesis: section 1, where the epithelial sheath is intact; section 2, where the epithelial sheath becomes fragmented; and section 3, where initial cellular cementogenesis begins. After fragmentation of the epithelial sheath, many keratin-positive epithelial sheath cells were embedded in the rapidly growing cellular cementum. A few unembedded epithelial cells located on the cementum surface. Dental follicle cells, precementoblasts, and cementoblasts showed immunoreactivity for vimentin and TNALP. In all three sections, there were virtually no cells possessing double immunoreactivity for vimentin-keratin or TNALP-keratin and only embedded epithelial cells showed TUNEL reactivity. Taken together, these findings suggest that: (1) epithelial sheath cells divide into two groups; one group is embedded in the cementum and thereafter dies by apoptosis, and the other survives on the cementum surface as epithelial cell rests of Malassez; and (2) epithelial sheath cells do not undergo epithelial-mesenchymal transition during initial cellular cementogenesis. PMID:26160988

Epithelial Cells in Urine: MedlinePlus Lab Test Information

MedlinePlus

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

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

PubMed

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

2016-08-01

The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, β-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland. © 2016 International Federation for Cell Biology.

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

PubMed

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

2018-06-01

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

IPF: new insight on pathogenesis and treatment.

PubMed

Harari, S; Caminati, A

2010-05-01

Recent years have seen a robust influx of exciting new observations regarding the mechanisms that regulate the initiation and progression of pulmonary fibrosis but the pathogenesis remains poorly understood. The search for an alternative hypothesis to unremitting, chronic inflammation as the primary explanation for the pathophysiology of idiopathic pulmonary fibrosis (IPF) derives, in part, from the lack of therapeutic efficacy of high-dose immunosuppressive therapy in patients with IPF. The inflammatory hypothesis of IPF has since been challenged by the epithelial injury hypothesis, in which fibrosis is believed to result from epithelial injury, activation, and/or apoptosis with abnormal wound healing. This hypothesis suggests that recurrent unknown injury to distal pulmonary parenchyma causes repeated epithelial injury and apoptosis. The resultant loss of alveolar epithelium exposes the underlying basement membrane to oxidative damage and degradation. Emerging concepts suggest that IPF is the result of epithelial-mesenchymal interaction. The initiation of this fibrotic response may depend upon genetic factors and environmental triggers; the role of Th1 or Th2 cell-derived cytokines may also be important. This process appears to be unique to usual interstitial pneumonia/IPF. It is clear that IPF is a heterogeneous disease with variations in pathology, high-resolution computed tomography findings, and patterns of progression. Idiopathic pulmonary fibrosis is a complex disorder, and no unifying hypothesis has been identified at present that explains all the abnormalities.

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

PubMed Central

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

2014-01-01

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

Material-induced Senescence (MIS): Fluidity Induces Senescent Type Cell Death of Lung Cancer Cells via Insulin-Like Growth Factor Binding Protein 5.

PubMed

Mano, Sharmy Saimon; Uto, Koichiro; Ebara, Mitsuhiro

2017-01-01

Objective: We propose here material-induced senescence (MIS) as a new therapeutic concept that limits cancer progression by stable cell cycle arrest. This study examined for the first time the effect of material fluidity on cellular senescence in lung carcinoma using poly(ε-caprolactone- co -D, L-lactide) (P(CL- co -DLLA)) with tunable elasticity and fluidity. Methods: The fluidity was varied by chemically crosslinking the polymer networks: the crosslinked P(CL- co -DLLA) shows solid-like properties with a stiffness of 260 kPa, while the non-crosslinked polymer exists in a quasi-liquid state with loss and storage moduli of 33 kPa and 11 kPa, respectively. Results: We found that cancer cells growing on the non-crosslinked, fluidic substrate undergo a non-apoptotic form of cell death and the cell cycle was accumulated in a G0/G1 phase. Next, we investigated the expression of biomarkers that are associated with cancer pathways. The cancer cells on the fluidic substrate expressed several biomarkers associated with senescence such as insulin-like growth factor binding protein 5 (IGFBP5). This result indicates that when cancer cells sense fluidity in their surroundings, the cells express IGFBP5, which in turn triggers the expression of tumor suppressor protein 53 and initiates cell cycle arrest at the G1 phase followed by cellular senescence. Furthermore, the cancer cells on the fluidic substrate maintained their epithelial phenotype, suggesting that the cancer cells do not undergo epithelial to mesenchymal transition. Conclusion: By considering these results as the fundamental information for MIS, our system could be applied to induce senescence in treatment-resistant cancers such as metastatic cancer or cancer stem cells.

Central Role of the NF-κB Pathway in the Scgb1a1-Expressing Epithelium in Mediating Respiratory Syncytial Virus-Induced Airway Inflammation.

PubMed

Tian, Bing; Yang, Jun; Zhao, Yingxin; Ivanciuc, Teodora; Sun, Hong; Wakamiya, Maki; Garofalo, Roberto P; Brasier, Allan R

2018-06-01

Lower respiratory tract infection with respiratory syncytial virus (RSV) produces profound inflammation. Despite an understanding of the role of adaptive immunity in RSV infection, the identity of the major sentinel cells initially triggering inflammation is controversial. Here we evaluate the role of nonciliated secretoglobin ( Scgb1a1 )-expressing bronchiolar epithelial cells in RSV infection. Mice expressing a tamoxifen (TMX)-inducible Cre recombinase-estrogen receptor fusion protein (CreERTM) knocked into the Scgb1a1 locus were crossed with mice that harbor a RelA conditional allele ( RelA fl ), with loxP sites flanking exons 5 to 8 of the Rel homology domain. The Scgb1a1 CreERTM/+ × RelA fl/fl mouse is a RelA conditional knockout (RelA CKO ) of a nonciliated epithelial cell population enriched in the small bronchioles. TMX-treated RelA CKO mice have reduced pulmonary neutrophilic infiltration and impaired expression and secretion of NF-κB-dependent cytokines in response to RSV. In addition, RelA CKO mice had reduced expression levels of interferon (IFN) regulatory factor 1/7 (IRF1/7) and retinoic acid-inducible gene I (RIG-I), components of the mucosal IFN positive-feedback loop. We demonstrate that RSV replication induces RelA to complex with bromodomain-containing protein 4 (BRD4), a cofactor required for RNA polymerase II (Pol II) phosphorylation, activating the atypical histone acetyltransferase (HAT) activity of BRD4 required for phospho-Ser2 Pol II formation, histone H3K122 acetylation, and cytokine secretion in vitro and in vivo TMX-treated RelA CKO mice have less weight loss and reduced airway obstruction/hyperreactivity yet similar levels of IFN-γ production despite higher levels of virus production. These data indicate that the nonciliated Scgb1a1 -expressing epithelium is a major innate sensor for restricting RSV infection by mediating neutrophilic inflammation and chemokine and mucosal IFN production via the RelA-BRD4 pathway. IMPORTANCE RSV infection is the most common cause of infant hospitalizations in the United States, resulting in 2.1 million children annually requiring medical attention. RSV primarily infects nasal epithelial cells, spreading distally to produce severe lower respiratory tract infections. Our study examines the role of a nonciliated respiratory epithelial cell population in RSV infection. We genetically engineered a mouse that can be selectively depleted of the NF-κB/RelA transcription factor in this subset of epithelial cells. These mice show an impaired activation of the bromodomain-containing protein 4 (BRD4) coactivator, resulting in reduced cytokine expression and neutrophilic inflammation. During the course of RSV infection, epithelial RelA-depleted mice have reduced disease scores and airway hyperreactivity yet increased levels of virus replication. We conclude that RelA-BRD4 signaling in nonciliated bronchiolar epithelial cells mediates neutrophilic airway inflammation and disease severity. This complex is an attractive target to reduce the severity of infection. Copyright © 2018 American Society for Microbiology.

Galectin-8 induces partial epithelial–mesenchymal transition with invasive tumorigenic capabilities involving a FAK/EGFR/proteasome pathway in Madin–Darby canine kidney cells

PubMed Central

Oyanadel, Claudia; Holmes, Christopher; Pardo, Evelyn; Retamal, Claudio; Shaughnessy, Ronan; Smith, Patricio; Cortés, Priscilla; Bravo-Zehnder, Marcela; Metz, Claudia; Feuerhake, Teo; Romero, Diego; Roa, Juan Carlos; Montecinos, Viviana; Soza, Andrea; González, Alfonso

2018-01-01

Epithelial cells can acquire invasive and tumorigenic capabilities through epithelial–mesenchymal-transition (EMT). The glycan-binding protein galectin-8 (Gal-8) activates selective β1-integrins involved in EMT and is overexpressed by certain carcinomas. Here we show that Gal-8 overexpression or exogenous addition promotes proliferation, migration, and invasion in nontumoral Madin–Darby canine kidney (MDCK) cells, involving focal-adhesion kinase (FAK)-mediated transactivation of the epidermal growth factor receptor (EGFR), likely triggered by α5β1integrin binding. Under subconfluent conditions, Gal-8–overexpressing MDCK cells (MDCK-Gal-8H) display hallmarks of EMT, including decreased E-cadherin and up-regulated expression of vimentin, fibronectin, and Snail, as well as increased β-catenin activity. Changes related to migration/invasion included higher expression of α5β1 integrin, extracellular matrix-degrading MMP13 and urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) protease systems. Gal-8–stimulated FAK/EGFR pathway leads to proteasome overactivity characteristic of cancer cells. Yet MDCK-Gal-8H cells still develop apical/basolateral polarity reverting EMT markers and proteasome activity under confluence. This is due to the opposite segregation of Gal-8 secretion (apical) and β1-integrins distribution (basolateral). Strikingly, MDCK-Gal-8H cells acquired tumorigenic potential, as reflected in anchorage-independent growth in soft agar and tumor generation in immunodeficient NSG mice. Therefore, Gal-8 can promote oncogenic-like transformation of epithelial cells through partial and reversible EMT, accompanied by higher proliferation, migration/invasion, and tumorigenic properties. PMID:29298841

Molecular mechanisms of ulcer healing.

PubMed

Tarnawski, A

2000-04-01

An ulcer in the gastrointestinal tract is a deep necrotic lesion penetrating the entire mucosal thickness and muscularis mucosae. Ulcer healing is an active process of filling the mucosal defect with proliferating and migrating epithelial and connective tissue cells. At the ulcer margin, epithelial cells proliferate and migrate onto the granulation tissue to cover (reepithelialize) the ulcer and also invade granulation tissue to reconstruct glandular structures within the ulcer scar. The reepithelialization and reconstruction of glandular structures is controlled by growth factors: trefoil peptides, EGF, HGF, bFGF and PDGF; and locally produced cytokines by regenerating cells in an orderly fashion and integrated manner to ensure the quality of mucosal restoration. These growth factors, most notably EGF, trigger cell proliferation via signal transduction pathways involving EGF-R, adapter proteins (Grb2, Shc and Sos), Ras, Raf1 and MAP (Erk1/Erk2) kinases, which, after translocation to nuclei, activate transcription factors and cell proliferation. Cell migration requires cytoskeletal rearrangements and is controlled by growth factors via Rho/Rac and signaling pathways involving PLC-gamma, PI-3 K and phosphorylation of focal adhesion proteins. Granulation tissue develops at the ulcer base. It consists of connective tissue cells: fibroblasts, macrophages and proliferating endothelial cells forming microvessels under the control of angiogenic growth factors: bFGF, VEGF and angiopoietins, which all promote angiogenesiscapillary vessel formation, essential for the restoration of microvascular network in the mucosa and thus crucial for oxygen and nutrient supply. The major mechanism of activation of angiogenic growth factors and their receptor expression appears to be hypoxia, which activates hypoxia-inducible factor, which binds to VEGF promoter.

Decoupling Internalization, Acidification and Phagosmal-Endosomal/Iysosomal Phagocytosis of Internalin A coated Beads in epithelial cells

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

Blanchette, C D; Woo, Y; Thomas, C

2008-12-22

Phagocytosis has been extensively examined in 'professional' phagocytic cells using pH sensitive dyes. However, in many of the previous studies, a separation between the end of internalization, beginning of acidification and completion of phagosomal-endosomal/lysosomal fusion was not clearly established, and in several cases, it was treated as a one-step process. In addition, very little work has been done to systematically examine phagosomal maturation in 'non-professional' phagocytic cells, such as epithelial cells. Therefore, in this study, we developed a simple and novel method to decouple and accurately measure particle internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in Madin-Darby Canine Kidney (MDCK) andmore » Caco-2 epithelial cells. Our method was developed using a pathogen mimetic system consisting of polystyrene beads coated with Internalin A (InlA), a membrane surface protein from Listeria monocytogenes known to trigger receptor-mediated internalization. We achieved independent measurements of the rates of internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in epithelial cells by combining the InlA-coated beads (InlA-beads) with antibody quenching, pH sensitive dyes and endosomal/lysosomal dyes, as follows: the rate of InlA bead internalization was measured via antibody quenching of a pH independent dye (Alexa488) conjugated to InlA-beads, the rate at which phagosomes containing internalized InlA beads became acidified was measured using a pH dependent dye (FITC) conjugated to the beads and the rate of phagosomal-endosomal/lysosomal fusion was measured using a combination of unlabeled InlA-beads and an endosomal/lysosomal dye. By performing these independent measurements under identical experimental conditions, we were able to decouple the three processes and establish time scales for each. In a separate set of experiments, we also exploited the phagosomal acidification process to demonstrate an additional, real31 time method for tracking bead binding, internalization and phagosomal acidification in both MDCK and Caco-2 cells, as well as 1 NIH 3T3 fibroblast cells, using FITC conjugated to InlA-beads or fibronectin-coated beads. Using this method, we found that the time scales for internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion were 23-32 min, 3-4 min and 74-120 min, respectively, for epithelial cells, MDCK and Caco-2, which are slower than the kinetics observed in professional phagocytes such as macrophages. Both the static and real-time methods developed here are expected to be readily and broadly applicable, as they simply require conjugation of a fluorophore to a pathogen or mimetic of interest in combination with common cell labeling dyes, and are not limited to the InlA ligand or cell types used here. As such, these methods hold promise for future measurements of receptor-mediated internalization in other cell systems, e.g. other pathogen-host systems.« less

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

PubMed

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

2005-04-01

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

Potential Role for a Carbohydrate Moiety in Anti-Candida Activity of Human Oral Epithelial Cells

PubMed Central

Steele, Chad; Leigh, Janet; Swoboda, Rolf; Ozenci, Hatice; Fidel, Paul L.

2001-01-01

Candida albicans is both a commensal and a pathogen at the oral mucosa. Although an intricate network of host defense mechanisms are expected for protection against oropharyngeal candidiasis, anti-Candida host defense mechanisms at the oral mucosa are poorly understood. Our laboratory recently showed that primary epithelial cells from human oral mucosa, as well as an oral epithelial cell line, inhibit the growth of blastoconidia and/or hyphal phases of several Candida species in vitro with a requirement for cell contact and with no demonstrable role for soluble factors. In the present study, we show that oral epithelial cell-mediated anti-Candida activity is resistant to gamma-irradiation and is not mediated by phagocytosis, nitric oxide, hydrogen peroxide, and superoxide oxidative inhibitory pathways or by nonoxidative components such as soluble defensin and calprotectin peptides. In contrast, epithelial cell-mediated anti-Candida activity was sensitive to heat, paraformaldehyde fixation, and detergents, but these treatments were accompanied by a significant loss in epithelial cell viability. Treatments that removed existing membrane protein or lipid moieties in the presence or absence of protein synthesis inhibitors had no effect on epithelial cell inhibitory activity. In contrast, the epithelial cell-mediated anti-Candida activity was abrogated after treatment of the epithelial cells with periodic acid, suggesting a role for carbohydrates. Adherence of C. albicans to oral epithelial cells was unaffected, indicating that the carbohydrate moiety is exclusively associated with the growth inhibition activity. Subsequent studies that evaluated specific membrane carbohydrate moieties, however, showed no role for sulfated polysaccharides, sialic acid residues, or glucose- and mannose-containing carbohydrates. These results suggest that oral epithelial cell-mediated anti-Candida activity occurs exclusively with viable epithelial cells through contact with C. albicans by an as-yet-undefined carbohydrate moiety. PMID:11598085

Thyroid Hormone-Induced Activation of Notch Signaling is Required for Adult Intestinal Stem Cell Development During Xenopus Laevis Metamorphosis.

PubMed

Hasebe, Takashi; Fujimoto, Kenta; Kajita, Mitsuko; Fu, Liezhen; Shi, Yun-Bo; Ishizuya-Oka, Atsuko

2017-04-01

In Xenopus laevis intestine during metamorphosis, the larval epithelial cells are removed by apoptosis, and the adult epithelial stem (AE) cells appear concomitantly. They proliferate and differentiate to form the adult epithelium (Ep). Thyroid hormone (TH) is well established to trigger this remodeling by regulating the expression of various genes including Notch receptor. To study the role of Notch signaling, we have analyzed the expression of its components, including the ligands (DLL and Jag), receptor (Notch), and targets (Hairy), in the metamorphosing intestine by real-time reverse transcription-polymerase chain reaction and in situ hybridization or immunohistochemistry. We show that they are up-regulated during both natural and TH-induced metamorphosis in a tissue-specific manner. Particularly, Hairy1 is specifically expressed in the AE cells. Moreover, up-regulation of Hairy1 and Hairy2b by TH was prevented by treating tadpoles with a γ-secretase inhibitor (GSI), which inhibits Notch signaling. More importantly, TH-induced up-regulation of LGR5, an adult intestinal stem cell marker, was suppressed by GSI treatment. Our results suggest that Notch signaling plays a role in stem cell development by regulating the expression of Hairy genes during intestinal remodeling. Furthermore, we show with organ culture experiments that prolonged exposure of tadpole intestine to TH plus GSI leads to hyperplasia of secretory cells and reduction of absorptive cells. Our findings here thus provide evidence for evolutionarily conserved role of Notch signaling in intestinal cell fate determination but more importantly reveal, for the first time, an important role of Notch pathway in the formation of adult intestinal stem cells during vertebrate development. Stem Cells 2017;35:1028-1039. © 2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Deregulation of SGK1 in Ulcerative Colitis: A Paradoxical Relationship Between Immune Cells and Colonic Epithelial Cells.

PubMed

Spagnuolo, Rocco; Dattilo, Vincenzo; D'Antona, Lucia; Cosco, Cristina; Tallerico, Rossana; Ventura, Valeria; Conforti, Francesco; Camastra, Caterina; Mancina, Rosellina M; Catalogna, Giada; Cosco, Vincenzo; Iuliano, Rodolfo; Carbone, Ennio; Perrotti, Nicola; Amato, Rosario; Doldo, Patrizia

2018-05-18

Inflammatory bowel disease (IBD) is due to the interaction of genetic and environmental factors that trigger an unbalanced immune response ultimately resulting in the peculiar inflammatory reaction. Experimental models of IBD point to a role of T-cell-derived cytokines (Th17) and to SGK1 as mediator of the Th17 switch. We hypothesize that SGK1, a salt inducible kinase, directs lymphocytic behavior and tissue damage. Eleven controls and 32 ulcerative colitis (UC) patients were randomized according to endoscopic Mayo score. Mucosal biopsies from different intestinal tracts were analyzed by immunohistochemistry and quantitative real-time polymerase chain reaction to check the expression of disease markers including SGK1. Peripheral blood mononuclear cells (PBMCs) from patients and controls were analyzed by fluorescence-activated cell sorting. Finally, an in vitro cell model was developed to test the hypothesis. SGK1 mRNA and protein expression in lesional areas of UC patients were lower than in normal peri-lesional areas of the same patients and in normal tissues of healthy controls. SGK1 expression was increased in PBMCs from UC patients, particularly in the CD4+ cell population, enriched in Th17 cells. IL17/IL13 was increased in patients and correlated with SGK1 expression. Genetically engineered Jurkat cells confirmed the effect of SGK1 overexpression on viability of RKO cells. These observations suggest a pathogenic mechanism whereby SGK1 overexpression in CD4+ T cells induces the secretion of the inflammatory cytokines IL17 and IL13, which downregulate the expression of SGK1 in target tissues. Our data suggest a novel hypothesis in the pathogenesis of UC, integrating colonic epithelial cells and lymphocytes.

Histogenesis of the epithelial component of rat thymus: an ultrastructural and immunohistological analysis.

PubMed

Vicente, A; Varas, A; Sacedón, R; Zapata, A G

1996-04-01

Despite the assumed importance of thymic cell microenvironments for governing T-cell maturation, little is known about the ontogeny of their cell components. A few studies have analyzed previously the ontogenetical development of rat thymic epithelium (Bogojevic et al. 1990. Period. Biol., 92:126; Kampinga and Aspinall 1990 Harwood Acad. Pub., London, pp. 149-186; Micic et al., 1991 Dev. Comp. Immunol., 15:443-450) and recently we have reported the development of both interdigitating/dendritic cells and macrophages (Vicente et al., 1994 Immunology, 82:75-81, 1995 Immunology, 85:99-105). In the present work we analyze in situ ultrastructural, immunohistochemical, and histoenzymatically the appearance and development of the thymic epithelial cell component in both embryonic and neonatal Wistar rats with special emphasis on the origin of the different epithelial cell types, the occurrence or absence of a common precursor for these, and the expression of MHC molecules. The thymic primordium of 13-day-old embryos is formed by a homogeneous population of primitive epithelial cells differentiating gradually into various epithelial cell subtypes of both the cortex and the medulla. In the cortex, subcapsular and stroma-supporting epithelial cells appear at days 14-15 as two structurally different cell entities. At the same time, stroma-supporting, keratinized, and vacuolated epithelial cells occur in the thymic medulla. These last two cell types differentiate subsequently into Hassall's bodies and hypertrophied cells. Lympho-epithelial cell complexes are identified in the deep cortex around birth, when the cortical parenchyma houses a transitional erythropoiesis. mAbs (His-39, RMC-20) which recognize medullary epithelial cells in the adult thymus stain positively cells of the thymic primordium as early as day 16 of embryonic life. Cortical epithelial cell markers (His-37, RMC-17) appear, however, slightly later and the subcapsulary region is not established until postnatal life. MHC class I and class II molecules can be identified on epithelial cells in the thymus of 15-day-old embryonic rats although they reach the highest expression around birth. Our results confirm the heterogeneity of the thymic epithelial component, the persistence of primitive, non-differentiated epithelial cells morphologically similar to those occurring in the early thymic primordium in adult thymus, and the mutual relevance of epithelial cells and thymocytes for an adequate development of rat thymus gland.

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

PubMed

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

2015-11-01

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

The world of epithelial sheets.

PubMed

Honda, Hisao

2017-06-01

An epithelium is a layer of closely connected cells covering the body or lining a body cavity. In this review, several fundamental questions are addressed regarding the epithelium. (i) While an epithelium functions as barrier against the external environment, how is barrier function maintained during its construction? (ii) What determines the apical and basal sides of epithelial layer? (iii) Is there any relationship between the apical side of the epithelium and the apical membrane of an epithelial cell? (iv) Why are hepatocytes (liver cells) called epithelial, even though they differ completely from column-like shape of typical epithelial cells? Keeping these questions in mind, multiple shapes of epithelia were considered, extracting a few of their elemental processes, and constructing a virtual world of epithelia by combining them. Epithelial cells were also classified into several types based on the number of apical domains of each cell. In addition, an intracellular organelle was introduced within epithelial cells, the vacuolar apical compartment (VAC), which is produced within epithelial cells surrounded by external cell matrix (ECM). The VAC interacts with areas of cell-cell contact of the cell surface membrane and is converted to apical membrane. The properties of VACs enable us to answer the initial questions posed above. Finally, the genetic and molecular mechanisms of epithelial morphogenesis are discussed. © 2017 Japanese Society of Developmental Biologists.

Palliative Care in Improving Quality of Life and Symptoms in Patients With Stage III-IV Pancreatic or Ovarian Cancer

ClinicalTrials.gov

2014-12-18

Recurrent Ovarian Epithelial Cancer; Recurrent Ovarian Germ Cell Tumor; Recurrent Pancreatic Cancer; Stage III Pancreatic Cancer; Stage IIIA Ovarian Epithelial Cancer; Stage IIIA Ovarian Germ Cell Tumor; Stage IIIB Ovarian Epithelial Cancer; Stage IIIB Ovarian Germ Cell Tumor; Stage IIIC Ovarian Epithelial Cancer; Stage IIIC Ovarian Germ Cell Tumor; Stage IV Ovarian Epithelial Cancer; Stage IV Ovarian Germ Cell Tumor; Stage IV Pancreatic Cancer

Thyroid Hormone‐Induced Activation of Notch Signaling is Required for Adult Intestinal Stem Cell Development During Xenopus Laevis Metamorphosis

PubMed Central

Fujimoto, Kenta; Kajita, Mitsuko; Fu, Liezhen; Shi, Yun‐Bo; Ishizuya‐Oka, Atsuko

2016-01-01

Abstract In Xenopus laevis intestine during metamorphosis, the larval epithelial cells are removed by apoptosis, and the adult epithelial stem (AE) cells appear concomitantly. They proliferate and differentiate to form the adult epithelium (Ep). Thyroid hormone (TH) is well established to trigger this remodeling by regulating the expression of various genes including Notch receptor. To study the role of Notch signaling, we have analyzed the expression of its components, including the ligands (DLL and Jag), receptor (Notch), and targets (Hairy), in the metamorphosing intestine by real‐time reverse transcription‐polymerase chain reaction and in situ hybridization or immunohistochemistry. We show that they are up‐regulated during both natural and TH‐induced metamorphosis in a tissue‐specific manner. Particularly, Hairy1 is specifically expressed in the AE cells. Moreover, up‐regulation of Hairy1 and Hairy2b by TH was prevented by treating tadpoles with a γ‐secretase inhibitor (GSI), which inhibits Notch signaling. More importantly, TH‐induced up‐regulation of LGR5, an adult intestinal stem cell marker, was suppressed by GSI treatment. Our results suggest that Notch signaling plays a role in stem cell development by regulating the expression of Hairy genes during intestinal remodeling. Furthermore, we show with organ culture experiments that prolonged exposure of tadpole intestine to TH plus GSI leads to hyperplasia of secretory cells and reduction of absorptive cells. Our findings here thus provide evidence for evolutionarily conserved role of Notch signaling in intestinal cell fate determination but more importantly reveal, for the first time, an important role of Notch pathway in the formation of adult intestinal stem cells during vertebrate development. Stem Cells 2017;35:1028–1039 PMID:27870267

Efficient Immortalization of Primary Nasopharyngeal Epithelial Cells for EBV Infection Study

PubMed Central

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

2013-01-01

Nasopharyngeal carcinoma (NPC) is common among southern Chinese including the ethnic Cantonese population living in Hong Kong. Epstein-Barr virus (EBV) infection is detected in all undifferentiated type of NPC in this endemic region. Establishment of stable and latent EBV infection in premalignant nasopharyngeal epithelial cells is an early event in NPC development and may contribute to its pathogenesis. Immortalized primary nasopharyngeal epithelial cells represent an important tool for investigation of EBV infection and its tumorigenic potential in this special type of epithelial cells. However, the limited availability and small sizes of nasopharyngeal biopsies have seriously restricted the establishment of primary nasopharyngeal epithelial cells for immortalization. A reliable and effective method to immortalize primary nasopharyngeal epithelial cells will provide unrestricted materials for EBV infection studies. An earlier study has reported that Bmi-1 expression could immortalize primary nasopharyngeal epithelial cells. However, its efficiency and actions in immortalization have not been fully characterized. Our studies showed that Bmi-1 expression alone has limited ability to immortalize primary nasopharyngeal epithelial cells and additional events are often required for its immortalization action. We have identified some of the key events associated with the immortalization of primary nasopharyngeal epithelial cells. Efficient immortalization of nasopharyngeal epithelial cells could be reproducibly and efficiently achieved by the combined actions of Bmi-1 expression, activation of telomerase and silencing of p16 gene. Activation of MAPK signaling and gene expression downstream of Bmi-1 were detected in the immortalized nasopharyngeal epithelial cells and may play a role in immortalization. Furthermore, these newly immortalized nasopharyngeal epithelial cells are susceptible to EBV infection and supported a type II latent EBV infection program characteristic of EBV-infected nasopharyngeal carcinoma. The establishment of an efficient method to immortalize primary nasopharyngeal epithelial cells will facilitate the investigation into the role of EBV infection in pathogenesis of nasopharyngeal carcinoma. PMID:24167620

Divergent Effects of Neutrophils on Fas-Induced Pulmonary Inflammation, Apoptosis, and Lung Damage.

PubMed

Bruns, Bastian; Hönle, Theresia; Kellermann, Philipp; Ayala, Alfred; Perl, Mario

2017-02-01

Pulmonary Fas activation is essential in the pathogenesis of the acute respiratory distress syndrome. It remains unclear whether Fas-induced lung injury is dependent on neutrophils or mainly triggered by epithelial cell apoptosis. The contribution of lung epithelial cells (LEC) and alveolar macrophages (AM) remains elusive.Mice were neutrophil reduced prior to intratracheal instillation of Fas-activating (Jo2) or isotype antibody for 6 or 18 h. LEC and AM were incubated with Jo2 and in the presence of nuclear factor kappa B, p-38 mitogen activated protein kinase (p38MAPK), or extracellular signal regulating kinase 1/2 (ERK1/2) inhibitors. Cytokines were assessed by cytometric bead array or ELISA. Apoptosis was quantified via active caspase-3 Western blotting and Terminal Deoxynucleotide Transferase dUTP Nick End Labeling (TUNEL). Lung injury was assessed by bronchoalveolar lavage fluid (BALF) protein concentration and lung histology.KC, IL-6, and MCP-1 were markedly increased in lung, plasma, and BALF 18 h after Jo2 in the presence of neutrophils; in neutrophil-reduced mice lungs, MCP-1, but not KC or IL-6, was even further enhanced. Six hours after Jo2, BALF protein was markedly increased only in the presence of neutrophils. Apoptosis remained unaffected by neutrophil reduction. AM released MCP-1 and underwent apoptosis at lower concentrations of Jo2 than LEC. Inhibition of p38MAPK significantly increased, while inhibition of ERK1/2 reduced AM and LEC apoptosis.In conclusion, neutrophils are a necessary component of Fas-induced lung damage, while not affecting lung apoptosis directly per se. LEC display higher resistance to Fas-triggered inflammation and apoptosis than AM.

Exocytosis of vacuolar apical compartment (VAC): a cell-cell contact controlled mechanism for the establishment of the apical plasma membrane domain in epithelial cells

PubMed Central

1988-01-01

The vacuolar apical compartment (VAC) is an organelle found in Madin- Darby canine kidney (MDCK) cells with incomplete intercellular contacts by incubation in 5 microM Ca++ and in cells without contacts (single cells in subconfluent culture); characteristically, it displays apical biochemical markers and microvilli and excludes basolateral markers (Vega-Salas, D. E., P. J. I. Salas, and E. Rodriguez-Boulan. 1987. J. Cell Biol. 104:1249-1259). The apical surface of cells kept under these culture conditions is immature, with reduced numbers of microvilli and decreased levels of an apical biochemical marker (184 kD), which is, however, still highly polarized (Vega-Salas, D. E., P. J. I. Salas, D. Gundersen, and E. Rodriguez-Boulan. 1987. J. Cell Biol. 104:905-916). We describe here the morphological stages of VAC exocytosis which ultimately lead to the establishment of a differentiated apical domain. Addition of 1.8 mM Ca++ to monolayers developed in 5 microM Ca++ causes the rapid (20-40 min) fusion of VACs with the plasma membrane and their accessibility to external antibodies, as demonstrated by immunofluorescence, immunoperoxidase EM, and RIA with antibodies against the 184-kD apical plasma membrane marker. Exocytosis occurs towards areas of cell-cell contact in the developing lateral surface where they form intercellular pockets; fusion images are always observed immediately adjacent to the incomplete junctional bands detected by the ZO-1 antibody (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766). Blocks of newly incorporated VAC microvilli and 184-kD protein progressively move from intercellular ("primitive" lateral) spaces towards the microvilli-poor free cell surface. The definitive lateral domain is sealed behind these blocks by the growing tight junctional fence. These results demonstrate a fundamental role of cell-cell contact-mediated VAC exocytosis in the establishment of epithelial surface polarity. Because isolated stages (intercellular pockets) of the stereotyped sequence of events triggered by the establishment of intercellular contacts in MDCK cells have been reported during normal differentiation of intestine epithelium (Colony, P. C., and M. R. Neutra. 1983. Dev. Biol. 97:349-363), we speculate that the mechanism we describe here plays an important role in the establishment of epithelial cell polarity in vivo. PMID:3053735

In vivo collective cell migration requires an LPAR2-dependent increase in tissue fluidity.

PubMed

Kuriyama, Sei; Theveneau, Eric; Benedetto, Alexandre; Parsons, Maddy; Tanaka, Masamitsu; Charras, Guillaume; Kabla, Alexandre; Mayor, Roberto

2014-07-07

Collective cell migration (CCM) and epithelial-mesenchymal transition (EMT) are common to cancer and morphogenesis, and are often considered to be mutually exclusive in spite of the fact that many cancer and embryonic cells that have gone through EMT still cooperate to migrate collectively. Here we use neural crest (NC) cells to address the question of how cells that have down-regulated cell-cell adhesions can migrate collectively. NC cell dissociation relies on a qualitative and quantitative change of the cadherin repertoire. We found that the level of cell-cell adhesion is precisely regulated by internalization of N-cadherin downstream of lysophosphatidic acid (LPA) receptor 2. Rather than promoting the generation of single, fully mesenchymal cells, this reduction of membrane N-cadherin only triggers a partial mesenchymal phenotype. This intermediate phenotype is characterized by an increase in tissue fluidity akin to a solid-like-to-fluid-like transition. This change of plasticity allows cells to migrate under physical constraints without abolishing cell cooperation required for collectiveness. © 2014 Kuriyama et al.

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

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

Terranova, Victor Paul

1979-01-01

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

Fourier transform infra-red spectroscopic signatures for lung cells' epithelial mesenchymal transition: A preliminary report

NASA Astrophysics Data System (ADS)

Sarkar, Atasi; Sengupta, Sanghamitra; Mukherjee, Anirban; Chatterjee, Jyotirmoy

2017-02-01

Infra red (IR) spectral characterization can provide label-free cellular metabolic signatures of normal and diseased circumstances in a rapid and non-invasive manner. Present study endeavoured to enlist Fourier transform infra red (FTIR) spectroscopic signatures for lung normal and cancer cells during chemically induced epithelial mesenchymal transition (EMT) for which global metabolic dimension is not well reported yet. Occurrence of EMT was validated with morphological and immunocytochemical confirmation. Pre-processed spectral data was analyzed using ANOVA and principal component analysis-linear discriminant analysis (PCA-LDA). Significant differences observed in peak area corresponding to biochemical fingerprint (900-1800 cm- 1) and high wave-number (2800-3800 cm- 1) regions contributed to adequate PCA-LDA segregation of cells undergoing EMT. The findings were validated by re-analysis of data using another in-house built binary classifier namely vector valued regularized kernel approximation (VVRKFA), in order to understand EMT progression. To improve the classification accuracy, forward feature selection (FFS) tool was employed in extracting potent spectral signatures by eliminating undesirable noise. Gradual increase in classification accuracy with EMT progression of both cell types indicated prominence of the biochemical alterations. Rapid changes in cellular metabolome noted in cancer cells within first 24 h of EMT induction along with higher classification accuracy for cancer cell groups in comparison to normal cells might be attributed to inherent differences between them. Spectral features were suggestive of EMT triggered changes in nucleic acid, protein, lipid and bound water contents which can emerge as the useful markers to capture EMT related cellular characteristics.

Expression and cytokine regulation of immune recognition elements by normal human biliary epithelial and established liver cell lines in vitro.

PubMed

Cruickshank, S M; Southgate, J; Selby, P J; Trejdosiewicz, L K

1998-10-01

Biliary epithelial cells are targets of immune-mediated attack in conditions such as primary biliary cirrhosis and allograft rejection. This has been attributed to the ability of biliary epithelial cells to express ligands for T cell receptors. We aimed to investigate the expression of immune recognition elements and the effects of pro-inflammatory and anti-inflammatory cytokines on cell surface phenotypes of normal human biliary epithelial cells and established human liver-derived (PLC/PRF/5, HepG2, Hep3B and CC-SW) lines. Cells were cultured in the presence or absence of cytokines for 72 h, and expression of cell surface molecules was assessed by flow cytometry and immunofluorescence. All cell lines expressed MHC class I, ICAM-1 (CD54), LFA-3 (CD58) and EGF receptor, and all but Hep3B expressed Fas/Apo-1 (CD95). Unlike hepatocyte-derived cell lines, biliary epithelial cells and CC-SW expressed CD40 and CD44. As expected, IFNgamma and TNFalpha upregulated expression of ICAM-1, MHC class I and MHC class II, particularly in biliary epithelial cells. TGFbeta downregulated these molecules and downregulated CD95 on biliary epithelial cells, but upregulated LFA-3. The Th2 cytokines had little effect, although IL-4 upregulated CD95 expression on biliary epithelial cells. IFNgamma upregulated CD40 expression on biliary epithelial cells, CC-SW and HepG2. These findings imply that biliary epithelial cells may be capable of interacting with activated T lymphocytes via CD40 and LFA-3, which are thought to be important T cell accessory ligands for T cell activation in a B7-independent manner. Sensitivity to pro-inflammatory cytokines and expression of CD95 may explain why biliary epithelial cells are primary targets for autoimmune attack.

Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors

PubMed Central

Akbay, Esra A; Koyama, Shohei; Carretero, Julian; Altabef, Abigail; Tchaicha, Jeremy H; Christensen, Camilla L; Mikse, Oliver R; Cherniack, Andrew D; Beauchamp, Ellen M; Pugh, Trevor J; Wilkerson, Matthew D; Fecci, Peter E; Butaney, Mohit; Reibel, Jacob B; Soucheray, Margaret; Cohoon, Travis J; Janne, Pasi A; Meyerson, Matthew; Hayes, D. Neil; Shapiro, Geoffrey I; Shimamura, Takeshi; Sholl, Lynette M; Rodig, Scott J; Freeman, Gordon J; Hammerman, Peter S; Dranoff, Glenn; Wong, Kwok-Kin

2013-01-01

The success in lung cancer therapy with Programmed Death (PD)-1 blockade suggests that immune escape mechanisms contribute to lung tumor pathogenesis. We identified a correlation between Epidermal Growth Factor Receptor (EGFR) pathway activation and a signature of immunosuppression manifested by upregulation of PD-1, PD-L1, cytotoxic T lymphocyte antigen-4 (CTLA-4), and multiple tumor-promoting inflammatory cytokines. We observed decreased cytotoxic T cells and increased markers of T cell exhaustion in mouse models of EGFR-driven lung cancer. PD-1 antibody blockade improved the survival of mice with EGFR-driven adenocarcinomas by enhancing effector T cell function and lowering the levels of tumor-promoting cytokines. Expression of mutant EGFR in bronchial epithelial cells induced PD-L1, and PD-L1 expression was reduced by EGFR inhibitors in non-small cell lung cancer cell lines with activated EGFR. These data suggest that oncogenic EGFR signaling remodels the tumor microenvironment to trigger immune escape, and mechanistically link treatment response to PD-1 inhibition. PMID:24078774

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

PubMed Central

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

2008-01-01

The nose is an attractive source of airway epithelial cells, particularly in populations in which bronchoscopy may not be possible. However, substituting nasal cells for bronchial epithelial cells in the study of airway inflammation depends upon comparability of responses, and evidence for this is lacking. Our objective was to determine whether nasal epithelial cell inflammatory mediator release and receptor expression reflect those of bronchial epithelial cells. Paired cultures of undifferentiated nasal and bronchial epithelial cells were obtained from brushings from 35 subjects, including 5 children. Cells were subject to morphologic and immunocytochemical assessment. Mediator release from resting and cytokine-stimulated cell monolayers was determined, as was cell surface receptor expression. Nasal and bronchial cells had identical epithelial morphology and uniform expression of cytokeratin 19. There were no differences in constitutive expression of CD44, intercellular adhesion molecule-1, αvβ3, and αvβ5. Despite significantly higher constitutive release of IL-8, IL-6, RANTES (regulated on activation, normal T cell expressed and secreted), and matrix metalloproteinase (MMP)-9 from nasal compared with bronchial cells, the increments in release of all studied mediators in response to stimulation with IL-1β and TNF-α were similar, and there were significant positive correlations between nasal and bronchial cell secretion of IL-6, RANTES, vascular endothelial growth factor, monocyte chemoattractant protein-1, MMP-9, and tissue inhibitor of metalloproteinase-1. Despite differences in absolute mediator levels, the responses of nasal and bronchial epithelial cells to cytokine stimulation were similar, expression of relevant surface receptors was comparable, and there were significant correlations between nasal and bronchial cell mediator release. Therefore, nasal epithelial cultures constitute an accessible surrogate for studying lower airway inflammation. PMID:18483420

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

PubMed

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

2008-11-01

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

Non-toxic engineered carbon nanodiamond concentrations induce oxidative/nitrosative stress, imbalance of energy metabolism, and mitochondrial dysfunction in microglial and alveolar basal epithelial cells.

PubMed

Fresta, Claudia G; Chakraborty, Aishik; Wijesinghe, Manjula B; Amorini, Angela M; Lazzarino, Giacomo; Lazzarino, Giuseppe; Tavazzi, Barbara; Lunte, Susan M; Caraci, Filippo; Dhar, Prajnaparamita; Caruso, Giuseppe

2018-02-14

Engineered nanoparticles are finding a wide spectrum of biomedical applications, including drug delivery and capacity to trigger cytotoxic phenomena, potentially useful against tumor cells. The full understanding of their biosafety and interactions with cell processes is mandatory. Using microglial (BV-2) and alveolar basal epithelial (A549) cells, in this study we determined the effects of engineered carbon nanodiamonds (ECNs) on cell viability, nitric oxide (NO) and reactive oxygen species (ROS) production, as well as on energy metabolism. Particularly, we initially measured decrease in cell viability as a function of increasing ECNs doses, finding similar cytotoxic ECN effects in the two cell lines. Subsequently, using apparently non-cytotoxic ECN concentrations (2 µg/mL causing decrease in cell number < 5%) we determined NO and ROS production, and measured the concentrations of compounds related to energy metabolism, mitochondrial functions, oxido-reductive reactions, and antioxidant defences. We found that in both cell lines non-cytotoxic ECN concentrations increased NO and ROS production with sustained oxidative/nitrosative stress, and caused energy metabolism imbalance (decrease in high energy phosphates and nicotinic coenzymes) and mitochondrial malfunctioning (decrease in ATP/ADP ratio).These results underline the importance to deeply investigate the molecular and biochemical changes occurring upon the interaction of ECNs (and nanoparticles in general) with living cells, even at apparently non-toxic concentration. Since the use of ECNs in biomedical field is attracting increasing attention the complete evaluation of their biosafety, toxicity and/or possible side effects both in vitro and in vivo is mandatory before these highly promising tools might find the correct application.

Androgen Receptor Expression in Epithelial and Stromal Cells of Prostatic Carcinoma and Benign Prostatic Hyperplasia.

PubMed

Filipovski, Vanja; Kubelka-Sabit, Katerina; Jasar, Dzengis; Janevska, Vesna

2017-08-15

Prostatic carcinoma (PCa) derives from prostatic epithelial cells. However stromal microenvironment, associated with malignant epithelium, also plays a role in prostatic carcinogenesis. Alterations in prostatic stromal cells contribute to the loss of growth control in epithelial cells that lead to progression of PCa. To analyse the differences between Androgen Receptor (AR) expression in both epithelial and stromal cells in PCa and the surrounding benign prostatic hyperplasia (BPH) and to compare the results with tumour grade. Samples from 70 cases of radical prostatectomy specimens were used. The expression and intensity of the signal for AR was analysed in the epithelial and stromal cells of PCa and BPH, and the data was quantified using histological score (H-score). AR showed significantly lower expression in both epithelial and stromal cells of PCa compared to BPH. In PCa a significant positive correlation of AR expression was found between stromal and epithelial cells of PCa. AR expression showed a correlation between the stromal cells of PCa and tumour grade. AR expression is reduced in epithelial and stromal cells of PCa. Expression of AR in stromal cells of PCa significantly correlates with tumour grade.

Akt recruits Dab2 to albumin endocytosis in the proximal tubule.

PubMed

Koral, Kelly; Li, Hui; Ganesh, Nandita; Birnbaum, Morris J; Hallows, Kenneth R; Erkan, Elif

2014-12-15

Proximal tubule epithelial cells have a highly sophisticated endocytic machinery to retrieve the albumin in the glomerular filtrate. The megalin-cubilin complex and the endocytic adaptor disabled-2 (Dab2) play a pivotal role in albumin endocytosis. We previously demonstrated that protein kinase B (Akt) regulates albumin endocytosis in the proximal tubule through an interaction with Dab2. Here, we examined the nature of Akt-Dab2 interaction. The pleckstrin homology (PH) and catalytic domains (CD) of Akt interacted with the proline-rich domain (PRD) of Dab2 based on yeast-two hybrid (Y2H) experiments. Pull-down experiments utilizing the truncated constructs of Dab2 demonstrated that the initial 11 amino acids of Dab2-PRD were sufficient to mediate the interaction between Akt and Dab2. Endocytosis experiments utilizing Akt1- and Akt2-silencing RNA revealed that both Akt1 and Akt2 mediate albumin endocytosis in proximal tubule epithelial cells; therefore, Akt1 and Akt2 may play a compensatory role in albumin endocytosis. Furthermore, both Akt isoforms phosphorylated Dab2 at Ser residues 448 and 449. Ser-to-Ala mutations of these Dab2 residues inhibited albumin endocytosis and resulted in a shift in location of Dab2 from the peripheral to the perinuclear area, suggesting the physiological relevance of these phosphorylation sites in albumin endocytosis. We conclude that both Akt1 and Akt2 are involved in albumin endocytosis, and phosphorylation of Dab2 by Akt induces albumin endocytosis in proximal tubule epithelial cells. Further delineation of how Akt affects expression/phosphorylation of endocytic adaptors and receptors will enhance our understanding of the molecular network triggered by albumin overload in the proximal tubule. Copyright © 2014 the American Physiological Society.

Scalloped and Yorkie are required for cell cycle re-entry of quiescent cells after tissue damage.

PubMed

Meserve, Joy H; Duronio, Robert J

2015-08-15

Regeneration of damaged tissues typically requires a population of active stem cells. How damaged tissue is regenerated in quiescent tissues lacking a stem cell population is less well understood. We used a genetic screen in the developing Drosophila melanogaster eye to investigate the mechanisms that trigger quiescent cells to re-enter the cell cycle and proliferate in response to tissue damage. We discovered that Hippo signaling regulates compensatory proliferation after extensive cell death in the developing eye. Scalloped and Yorkie, transcriptional effectors of the Hippo pathway, drive Cyclin E expression to induce cell cycle re-entry in cells that normally remain quiescent in the absence of damage. Ajuba, an upstream regulator of Hippo signaling that functions as a sensor of epithelial integrity, is also required for cell cycle re-entry. Thus, in addition to its well-established role in modulating proliferation during periods of tissue growth, Hippo signaling maintains homeostasis by regulating quiescent cell populations affected by tissue damage. © 2015. Published by The Company of Biologists Ltd.

Ex vivo gut culture for studying differentiation and migration of small intestinal epithelial cells

PubMed Central

Fu, Xing; Du, Min

2018-01-01

Epithelial cultures are commonly used for studying gut health. However, due to the absence of mesenchymal cells and gut structure, epithelial culture systems including recently developed three-dimensional organoid culture cannot accurately represent in vivo gut development, which requires intense cross-regulation of the epithelial layer with the underlying mesenchymal tissue. In addition, organoid culture is costly. To overcome this, a new culture system was developed using mouse embryonic small intestine. Cultured intestine showed spontaneous peristalsis, indicating the maintenance of the normal gut physiological structure. During 10 days of ex vivo culture, epithelial cells moved along the gut surface and differentiated into different epithelial cell types, including enterocytes, Paneth cells, goblet cells and enteroendocrine cells. We further used the established ex vivo system to examine the role of AMP-activated protein kinase (AMPK) on gut epithelial health. Tamoxifen-induced AMPKα1 knockout vastly impaired epithelial migration and differentiation of the developing ex vivo gut, showing the crucial regulatory function of AMPK α1 in intestinal health. PMID:29643147

Telomerase Activity Impacts on Epstein-Barr Virus Infection of AGS Cells

PubMed Central

Rac, Jürgen; Haas, Florian; Schumacher, Andrina; Middeldorp, Jaap M.; Delecluse, Henri-Jacques; Speck, Roberto F.

2015-01-01

The Epstein-Barr virus (EBV) is transmitted from host-to-host via saliva and is associated with epithelial malignancies including nasopharyngeal carcinoma (NPC) and some forms of gastric carcinoma (GC). Nevertheless, EBV does not transform epithelial cells in vitro where it is rapidly lost from infected primary epithelial cells or epithelial tumor cells. Long-term infection by EBV, however, can be established in hTERT-immortalized nasopharyngeal epithelial cells. Here, we hypothesized that increased telomerase activity in epithelial cells enhances their susceptibility to infection by EBV. Using HONE-1, AGS and HEK293 cells we generated epithelial model cell lines with increased or suppressed telomerase activity by stable ectopic expression of hTERT or of a catalytically inactive, dominant negative hTERT mutant. Infection experiments with recombinant prototypic EBV (rB95.8), recombinant NPC EBV (rM81) with increased epithelial cell tropism compared to B95.8, or recombinant B95.8 EBV with BZLF1-knockout that is not able to undergo lytic replication, revealed that infection frequencies positively correlate with telomerase activity in AGS cells but also partly depend on the cellular background. AGS cells with increased telomerase activity showed increased expression mainly of latent EBV genes, suggesting that increased telomerase activity directly acts on the EBV infection of epithelial cells by facilitating latent EBV gene expression early upon virus inoculation. Thus, our results indicate that infection of epithelial cells by EBV is a very selective process involving, among others, telomerase activity and cellular background to allow for optimized host-to-host transmission via saliva. PMID:25856387

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

PubMed

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

2013-09-24

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

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

PubMed

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

2008-05-16

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

Non-Saccharomyces yeasts protect against epithelial cell barrier disruption induced by Salmonella enterica subsp. enterica serovar Typhimurium.

PubMed

Smith, I M; Baker, A; Arneborg, N; Jespersen, L

2015-11-01

The human gastrointestinal epithelium makes up the largest barrier separating the body from the external environment. Whereas invasive pathogens cause epithelial barrier disruption, probiotic micro-organisms modulate tight junction regulation and improve epithelial barrier function. In addition, probiotic strains may be able to reduce epithelial barrier disruption caused by pathogenic species. The aim of this study was to explore non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Benchmarking against established probiotic strains, we evaluated the ability of four nonpathogenic yeast species to modulate transepithelial electrical resistance (TER) across a monolayer of differentiated human colonocytes (Caco-2 cells). Further, we assessed yeast modulation of a Salmonella Typhimurium-induced epithelial cell barrier function insult. Our findings demonstrate distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function. While the established probiotic yeast Saccharomyces boulardii increased TER across a Caco-2 monolayer by 30%, Kluyveromyces marxianus exhibited significantly stronger properties of TER enhancement (50% TER increase). In addition, our data demonstrate significant yeast-mediated modulation of Salmonella-induced epithelial cell barrier disruption and identify K. marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. This study demonstrates distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Further, our data demonstrate significant yeast-mediated modulation of Salmonella Typhimurium-induced epithelial cell barrier disruption and identify Kluyveromyces marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. This study is the first to demonstrate significant non-Saccharomyces yeast-mediated epithelial cell barrier protection from Salmonella invasion, thus encouraging future efforts aimed at confirming the observed effects in vivo and driving further strain development towards novel yeast probiotics. © 2015 The Society for Applied Microbiology.

Y-27632, a ROCK Inhibitor, Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing.

PubMed

Sun, Chi-Chin; Chiu, Hsiao-Ting; Lin, Yi-Fang; Lee, Kuo-Ying; Pang, Jong-Hwei Su

2015-01-01

Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency.

Overexpression of miR-183/-96/-182 triggers neuronal cell fate in Human Retinal Pigment Epithelial (hRPE) cells in culture.

PubMed

Davari, Maliheh; Soheili, Zahra-Soheila; Samiei, Shahram; Sharifi, Zohreh; Pirmardan, Ehsan Ranaei

2017-01-29

miR-183 cluster, composed of miR-183/-96/-182 genes, is highly expressed in the adult retina, particularly in photoreceptors. It involves in development, maturation and normal function of neuroretina. Ectopic overexpression of miR-183/-96/-182 genes was performed to assess reprogramming of hRPE cells. They were amplified from genomic DNA and cloned independently or in tandem configuration into pAAV.MCS vector. hRPE cells were then transfected with the recombinant constructs. Real-Time PCR was performed to measure the expression levels of miR-183/-96/-182 and that of several retina-specific neuronal genes such as OTX2, NRL, PDC and DCT. The transfected cells also were immunocytochemically examined for retina-specific neuronal markers, including Rhodopsin, red opsin, CRX, Thy1, CD73, recoverin and PKCα, to determine the cellular fate of the transfected hRPE cells. Data showed that upon miR-183/-96/-182 overexpression in hRPE cultures, the expression of neuronal genes including OTX2, NRL, PDC and DCT was also upregulated. Moreover, miR-183 cluster-treated hRPE cells were immunoreactive for neuronal markers such as Rhodopsin, red opsin, CRX and Thy1. Both transcriptional and translational upregulation of neuronal genes in miR-183 cluster-treated hRPE cells suggests that in vitro overexpression of miR-183 cluster could trigger reprogramming of hRPE cells to retinal neuron fate. Copyright © 2016 Elsevier Inc. All rights reserved.

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

PubMed

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

2015-05-01

Ciliary neurotrophic factor (CNTF), a well-known neuroprotective cytokine, has been found to play an important role in neurogenesis and functional regulations of neural stem cells. As one of the most innervated tissue, however, the role of CNTF in cornea epithelium remains unclear. This study was to explore the roles and mechanisms of CNTF in the activation of corneal epithelial stem/progenitor cells and wound healing of both normal and diabetic mouse corneal epithelium. In mice subjecting to mechanical removal of corneal epithelium, the corneal epithelial stem/progenitor cell activation and wound healing were promoted by exogenous CNTF application, while delayed by CNTF neutralizing antibody. In cultured corneal epithelial stem/progenitor cells, CNTF enhanced the colony-forming efficiency, stimulated the mitogenic proliferation, and upregulated the expression levels of corneal epithelial stem/progenitor cell-associated transcription factors. Furthermore, the promotion of CNTF on the corneal epithelial stem/progenitor cell activation and wound healing was mediated by the activation of STAT3. Moreover, in diabetic mice, the content of CNTF in corneal epithelium decreased significantly when compared with that of normal mice, and the supplement of CNTF promoted the diabetic corneal epithelial wound healing, accompanied with the advanced activation of corneal epithelial stem/progenitor cells and the regeneration of corneal nerve fibers. Thus, the capability of expanding corneal epithelial stem/progenitor cells and promoting corneal epithelial wound healing and nerve regeneration indicates the potential application of CNTF in ameliorating limbal stem cell deficiency and treating diabetic keratopathy. © 2014 AlphaMed Press.

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

PubMed Central

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

2017-01-01

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

Cystic fibrosis epithelial cells are primed for apoptosis as a result of increased Fas (CD95).

PubMed

Chen, Qiwei; Pandi, Sudha Priya Soundara; Kerrigan, Lauren; McElvaney, Noel G; Greene, Catherine M; Elborn, J Stuart; Taggart, Clifford C; Weldon, Sinéad

2018-02-24

Previous work suggests that apoptosis is dysfunctional in cystic fibrosis (CF) airways with conflicting results. We evaluated the relationship between dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) and apoptosis in CF airway epithelial cells. Apoptosis and associated caspase activity were analysed in non-CF and CF tracheal and bronchial epithelial cell lines. Basal levels of apoptosis and activity of caspase-3 and caspase-8 were significantly increased in CF epithelial cells compared to controls, suggesting involvement of extrinsic apoptosis signalling, which is mediated by the activation of death receptors, such as Fas (CD95). Increased levels of Fas were observed in CF epithelial cells and bronchial brushings from CF patients compared to non-CF controls. Neutralisation of Fas significantly inhibited caspase-3 activity in CF epithelial cells compared to untreated cells. In addition, activation of Fas significantly increased caspase-3 activity and apoptosis in CF epithelial cells compared to control cells. Overall, these results suggest that CF airway epithelial cells are more sensitive to apoptosis via increased levels of Fas and subsequent activation of the Fas death receptor pathway, which may be associated with dysfunctional CFTR. Copyright © 2018 European Cystic Fibrosis Society. All rights reserved.

Proliferation of epithelial cell rests, formation of apical cysts, and regression of apical cysts after periapical wound healing.

PubMed

Lin, Louis M; Huang, George T-J; Rosenberg, Paul A

2007-08-01

There is continuing controversy regarding the potential for inflammatory apical cysts to heal after nonsurgical endodontic therapy. Molecular cell biology may provide answers to a series of related questions. How are the epithelial cell rests of Malassez stimulated to proliferate? How are the apical cysts formed? How does the lining epithelium of apical cysts regress after endodontic therapy? Epithelial cell rests are induced to divide and proliferate by inflammatory mediators, proinflammatory cytokines, and growth factors released from host cells during periradicular inflammation. Quiescent epithelial cell rests can behave like restricted-potential stem cells if stimulated to proliferate. Formation of apical cysts is most likely caused by the merging of proliferating epithelial strands from all directions to form a three-dimensional ball mass. After endodontic therapy, epithelial cells in epithelial strands of periapical granulomas and the lining epithelium of apical cysts may stop proliferating because of a reduction in inflammatory mediators, proinflammatory cytokines, and growth factors. Epithelial cells will also regress because of activation of apoptosis or programmed cell death through deprivation of survival factors or by receiving death signals during periapical wound healing.

Synthetic Lethality of a Novel Small Molecule Against Mutant KRAS-Expressing Cancer Cells Involves AKT-Dependent ROS Production.

PubMed

Iskandar, Kartini; Rezlan, Majidah; Yadav, Sanjiv Kumar; Foo, Chuan Han Jonathan; Sethi, Gautam; Qiang, Yu; Bellot, Gregory L; Pervaiz, Shazib

2016-05-10

We recently reported the death-inducing activity of a small-molecule compound, C1, which triggered reactive oxygen species (ROS)-dependent autophagy-associated apoptosis in a variety of human cancer cell lines. In this study, we examine the ability of the compound to specifically target cancer cells harboring mutant KRAS with minimal activity against wild-type (WT) RAS-expressing cells. HCT116 cells expressing mutated KRAS are susceptible, while the WT-expressing HT29 cells are resistant. Interestingly, C1 triggers activation of mutant RAS, which results in the downstream phosphorylation and activation of AKT/PKB. Gene knockdown of KRAS or AKT or their pharmacological inhibition resulted in the abrogation of C1-induced ROS production and rescued tumor colony-forming ability. We also made use of HCT116 mutant KRAS knockout (KO) cells, which express only a single WT KRAS allele. Exposure of KO cells to C1 failed to increase mitochondrial ROS and cell death, unlike the parental cells harboring mutant KRAS. Similarly, mutant KRAS-transformed prostate epithelial cells (RWPE-1-RAS) were more sensitive to the ROS-producing and death-inducing effects of C1 than the vector only expressing RWPE-1 cells. An in vivo model of xenograft tumors generated with HCT116 KRAS(WT/MUT) or KRAS(WT/-) cells showed the efficacy of C1 treatment and its ability to affect the relative mitotic index in tumors harboring KRAS mutant. These data indicate a synthetic lethal effect against cells carrying mutant KRAS, which could have therapeutic implications given the paucity of KRAS-specific chemotherapeutic strategies. Antioxid. Redox Signal. 24, 781-794.

Mesenchymal precursor cells maintain the differentiation and proliferation potentials of breast epithelial cells

PubMed Central

2014-01-01

Introduction Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. Not surprisingly, aberrant stromal-epithelial interactions contribute to malignancies. Studies of the cellular and molecular mechanisms underlying these interactions require ex vivo experimental model systems that recapitulate the complexity of human tissue without compromising the differentiation and proliferation potentials of human primary cells. Methods We isolated and characterized human breast epithelial and mesenchymal precursors from reduction mammoplasty tissue and tagged them with lentiviral vectors. We assembled heterotypic co-cultures and compared mesenchymal and epithelial cells to cells in corresponding monocultures by analyzing growth, differentiation potentials, and gene expression profiles. Results We show that heterotypic culture of non-immortalized human primary breast epithelial and mesenchymal precursors maintains their proliferation and differentiation potentials and constrains their growth. We further describe the gene expression profiles of stromal and epithelial cells in co-cultures and monocultures and show increased expression of the tumor growth factor beta (TGFβ) family member inhibin beta A (INHBA) in mesenchymal cells grown as co-cultures compared with monocultures. Notably, overexpression of INHBA in mesenchymal cells increases colony formation potential of epithelial cells, suggesting that it contributes to the dynamic reciprocity between breast mesenchymal and epithelial cells. Conclusions The described heterotypic co-culture system will prove useful for further characterization of the molecular mechanisms mediating interactions between human normal or neoplastic breast epithelial cells and the stroma, and will provide a framework to test the relevance of the ever-increasing number of oncogenomic alterations identified in human breast cancer. PMID:24916766

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

PubMed

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

2002-10-01

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

Documentation of angiotensin II receptors in glomerular epithelial cells

NASA Technical Reports Server (NTRS)

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

1998-01-01

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

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

PubMed Central

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

2012-01-01

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

p53 activation by Ni(II) is a HIF-1α independent response causing caspases 9/3-mediated apoptosis in human lung cells

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

Wong, Victor C.; Morse, Jessica L.; Zhitkovich, Anatoly, E-mail: anatoly_zhitkovich@brown.edu

2013-06-15

Hypoxia mimic nickel(II) is a human respiratory carcinogen with a suspected epigenetic mode of action. We examined whether Ni(II) elicits a toxicologically significant activation of the tumor suppressor p53, which is typically associated with genotoxic responses. We found that treatments of H460 human lung epithelial cells with NiCl{sub 2} caused activating phosphorylation at p53-Ser15, accumulation of p53 protein and depletion of its inhibitor MDM4 (HDMX). Confirming the activation of p53, its knockdown suppressed the ability of Ni(II) to upregulate MDM2 and p21 (CDKN1A). Unlike DNA damage, induction of GADD45A by Ni(II) was p53-independent. Ni(II) also increased p53-Ser15 phosphorylation and p21more » expression in normal human lung fibroblasts. Although Ni(II)-induced stabilization of HIF-1α occurred earlier, it had no effect on p53 accumulation and Ser15 phosphorylation. Ni(II)-treated H460 cells showed no evidence of necrosis and their apoptosis and clonogenic death were suppressed by p53 knockdown. The apoptotic role of p53 involved a transcription-dependent program triggering the initiator caspase 9 and its downstream executioner caspase 3. Two most prominently upregulated proapoptotic genes by Ni(II) were PUMA and NOXA but only PUMA induction required p53. Knockdown of p53 also led to derepression of antiapoptotic MCL1 in Ni(II)-treated cells. Overall, our results indicate that p53 plays a major role in apoptotic death of human lung cells by Ni(II). Chronic exposure to Ni(II) may promote selection of resistant cells with inactivated p53, providing an explanation for the origin of p53 mutations by this epigenetic carcinogen. - Highlights: • Ni(II) is a strong activator of the transcription factor p53. • Apoptosis is a principal form of death by Ni(II) in human lung epithelial cells. • Ni(II)-activated p53 triggers caspases 9/3-mediated apoptotic program. • NOXA and PUMA are two main proapoptotic genes induced by Ni(II). • HIF-1α and p53 are independent stress responses to hypoxia-mimicking Ni(II)« less

Langerhans cells from human oral epithelium are more effective at stimulating allogeneic T cells in vitro than Langerhans cells from skin.

PubMed

Hasséus, B; Jontell, M; Bergenholtz, G; Dahlgren, U I

2004-06-01

This report is focused on the functional capacity of Langerhans cells (LC) in the epithelium of skin and oral mucosa, which both meet different antigenic challenges. The capacity of LC from human oral and skin epithelium to provide co-stimulatory signals to T cells in vitro was compared. LC in a crude suspension of oral epithelial cells had a significantly enhanced T cell co-stimulatory capacity compared to skin epithelial cells. This applied both to cultures with concanavalin A (con-A)-stimulated syngeneic T cells and to a mixed epithelial cell lymphocyte reaction involving allogeneic T cells. The co-stimulatory capacity of oral and skin epithelial cells was reduced by >70% if monoclonal antibodies against HLA-DR, -DP and -DQ were added to the cultures with allogeneic T cells, indicating the involvement of HLA class II expressing LC. Immunohistochemistry revealed that 6% of the epithelial cells were CD1a + LC in sections from both oral and skin epithelium. Interleukin (IL)-8 production was higher in cultures of oral epithelial cells and con-A stimulated T cells than in corresponding cultures with skin epithelial cells as accessory cells. The results suggest that LC in human oral epithelium are more efficient at stimulating T cells than those of skin.

Trichomonas vaginalis Contact-Dependent Cytolysis of Epithelial Cells

PubMed Central

Lustig, Gila; Ryan, Christopher M.; Secor, W. Evan

2013-01-01

Trichomonas vaginalis is an extracellular protozoan parasite that binds to the epithelium of the human urogenital tract during infection. In this study, we examined the propensities of 26 T. vaginalis strains to bind to and lyse prostate (BPH-1) and ectocervical (Ect1) epithelium and to lyse red blood cells (RBCs). We found that only three of the strains had a statistically significant preference for either BPH-1 (MSA1103) or Ect1 (LA1 and MSA1123). Overall, we observed that levels of adherence are highly variable among strains, with a 12-fold range of adherence on Ect1 cells and a 45-fold range on BPH-1 cells. Cytolysis levels displayed even greater variability, from no detectable cytolysis to 80% or 90% cytolysis of Ect1 and BPH-1, respectively. Levels of adherence and cytolysis correlate for weakly adherent/cytolytic strains, and a threshold of attachment was found to be necessary to trigger cytolysis; however, this threshold can be reached without inducing cytolysis. Furthermore, cytolysis was completely blocked when we prevented attachment of the parasites to host cells while allowing soluble factors complete access. We demonstrate that hemolysis was a rare trait, with only 4 of the 26 strains capable of lysing >20% RBCs with a 1:30 parasite/RBC ratio. Hemolysis also did not correlate with adherence to or cytolysis of either male (BPH-1)- or female (Ect1)-derived epithelial cell lines. Our results reveal that despite a broad range of pathogenic properties among different T. vaginalis strains, all strains show strict contact-dependent cytolysis. PMID:23429535

Exopolysaccharides from Lactobacillus delbrueckii OLL1073R-1 modulate innate antiviral immune response in porcine intestinal epithelial cells.

PubMed

Kanmani, Paulraj; Albarracin, Leonardo; Kobayashi, Hisakazu; Iida, Hikaru; Komatsu, Ryoya; Humayun Kober, A K M; Ikeda-Ohtsubo, Wakako; Suda, Yoshihito; Aso, Hisashi; Makino, Seiya; Kano, Hiroshi; Saito, Tadao; Villena, Julio; Kitazawa, Haruki

2018-01-01

Previous studies demonstrated that the extracellular polysaccharides (EPSs) produced by Lactobacillus delbrueckii OLL1073R-1 (LDR-1) improve antiviral immunity, especially in the systemic and respiratory compartments. However, it was not studied before whether those EPSs are able to beneficially modulate intestinal antiviral immunity. In addition, LDR-1-host interaction has been evaluated mainly with immune cells while its interaction with intestinal epithelial cells (IECs) was not addressed before. In this work, we investigated the capacity of EPSs from LDR-1 to modulate the response of porcine IECs (PIE cells) to the stimulation with the Toll-like receptor (TLR)-3 agonist poly(I:C) and the role of TLR2, TLR4, and TLR negative regulators in the immunoregulatory effect. We showed that innate immune response triggered by TLR3 activation in porcine IECs was differentially modulated by EPS from LDR-1. EPSs treatment induced an increment in the expression of interferon (IFN)-α and IFN-β in PIE cells after the stimulation with poly(I:C) as well as the expression of the antiviral factors MxA and RNase L. Those effects were related to the reduced expression of A20 in EPS-treated PIE cells. EPS from LDR-1 was also able to reduce the expression of IL-6 and proinflammatory chemokines. Although further in vivo studies are needed, our results suggest that these EPSs or a yogurt fermented with LDR-1 have potential to improve intestinal innate antiviral response and protect against intestinal viruses. Copyright © 2017 Elsevier Ltd. All rights reserved.

Inhibitory effects of clotrimazole on TNF-alpha-induced adhesion molecule expression and angiogenesis.

PubMed

Thapa, Dinesh; Lee, Jong Suk; Park, Min-A; Cho, Mi-Yeon; Park, Young-Joon; Choi, Han Gon; Jeong, Tae Cheon; Kim, Jung-Ae

2009-04-01

Cell adhesion molecules play a pivotal role in chronic inflammation and pathological angiogenesis. In the present study, we investigated the inhibitory effects of clotrimazole (CLT) on tumor necrosis factor (TNF)-alpha-induced changes in adhesion molecule expression. CLT dose-dependently inhibited monocyte chemoattractant protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) expressions in TNF-alpha-stimulated HT29 colonic epithelial cells. This inhibitory action of CLT correlated with a significant reduction in TNF-alpha-induced adhesion of monocytes to HT29 cells, which was comparable to the inhibitory effects of anti-ICAM-1 and VCAM-1 monoclonal antibodies on monocyte-epithelial adhesion. These inhibitory actions of CLT were, at least in part, attributable to the inhibition of redox sensitive NF-kappaB activation, as CLT inhibited TNF-alpha-induced ROS generation as well as NF-kappaB nuclear translocation and activation in HT29 cells. Furthermore, the inhibition of TNF-alpha-induced monocyte adhesion was also mimicked by the specific NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC). Inflammatory mediators including TNF-alpha have known to promote angiogenesis, which in turn further contributes to inflammatory pathology. Therefore, we additionally evaluated whether CLT modulates TNF-alpha-induced angiogenesis using in vivo chick chorioallantoic membrane (CAM) assay. The CAM assay showed that CLT dose-dependently attenuated TNF-alpha-induced angiogenesis, and the effect was correlated with decreased inflammation of the CAM tissue. In conclusion, our results suggest that CLT can inhibit TNF-alpha-triggered expression of adhesion molecules, ICAM-1 and VCAM-1, and angiogenesis during inflammation.

Characterization of newly established bovine intestinal epithelial cell line.

PubMed

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

2010-01-01

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

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

PubMed

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

2015-02-01

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

Aberrant c-erbB2 expression in cell clusters overlying focally disrupted breast myoepithelial cell layers: a trigger or sign for emergence of more aggressive cell clones?

PubMed Central

Zhang, Xichen; Hashemi, Shahreyar Shar; Yousefi, Morvarid; Ni, Jinsong; Wang, Qiuyue; Gao, Ling; Gong, Pengtao; Gao, Chunling; Sheng, Joy; Mason, Jeffrey; Man, Yan-gao

2008-01-01

Our recent studies revealed that cell clusters overlying focal myoepithelial cell layer disruption (FMCLD) had a significantly higher frequency of genetic instabilities and expression of invasion-related genes than their adjacent counterparts within the same duct. Our current study attempted to assess whether these cell clusters would also have elevated c-erbB2 expression. Human breast tumors (n=50) with a high frequency of FMCLD were analyzed with double immunohistochemistry, real-time RT-PCR, and chromogenic in situ hybridization for c-erbB2 protein and gene expression. Of 448 FMCLD detected, 404 (90.2%) were associated with cell clusters that had intense c-erbB2 immunoreactivities primarily in their cytoplasm, in contrast to their adjacent counterparts within the same duct, which had no or barely detectable c-erbB2 expression. These c-erbB2 positive cells were arranged as tongue-like projections, “puncturing” into the stroma, and about 20% of them were in direct continuity with tube-like structures that resembled blood vessels. Aberrant c-erbB2 expression was also seen in clusters of architecturally normal-appearing ducts that had distinct cytological abnormalities in both ME and epithelial cells, whereas not in their clear-cut normal counterparts. Molecular assays detected markedly higher c-erbB2 mRNA and gene amplification in cell clusters associated with FMCLD than in those associated with non-disrupted ME cell layers. Our findings suggest that cell clusters overlying FMCLD may represent the precursors of pending invasive lesions, and that aberrant cerbB2 expression may trigger or signify the emergence of biologically more aggressive cell clones. PMID:18726004

P16INK4a MEDIATED SUPPRESSION OF TELOMERASE IN NORMAL AND MALIGNANT HUMAN BREAST CELLS

PubMed Central

Bazarov, Alexey V.; van Sluis, Marjolein; Hines, Curtis; Bassett, Ekaterina; Beliveau, Alain; Campeau, Eric; Mukhopadhyay, Rituparna; Lee, Won Jae; Melodyev, Sonya; Zaslavsky, Yuri; Lee, Leonard; Rodier, Francis; Chicas, Agustin; Lowe, Scott W.; Benhattar, Jean; Ren, Bing; Campisi, Judith; Yaswen, Paul

2010-01-01

Summary The cyclin-dependent kinase inhibitor p16INK4a (CDKN2A) is an important tumor-suppressor gene frequently inactivated in human tumors. p16 suppresses the development of cancer by triggering an irreversible arrest of cell proliferation termed cellular senescence. Here, we describe another anti-oncogenic function of p16 in addition to its ability to halt cell cycle progression. We show that transient expression of p16 stably represses the hTERT gene, encoding the catalytic subunit of telomerase, in both normal and malignant breast epithelial cells. Short-term p16 expression increases the amount of histone H3 trimethylated on lysine 27 (H3K27) bound to the hTERT promoter, resulting in transcriptional silencing, likely mediated by polycomb complexes. Our results indicate that transient p16 exposure may prevent malignant progression in dividing cells by irreversible repression of genes, such as hTERT, whose activity is necessary for extensive self-renewal. PMID:20569236

Herpesvirus Entry into Host Cells Mediated by Endosomal Low pH.

PubMed

Nicola, Anthony V

2016-09-01

Herpesviral pathogenesis stems from infection of multiple cell types including the site of latency and cells that support lytic replication. Herpesviruses utilize distinct cellular pathways, including low pH endocytic pathways, to enter different pathophysiologically relevant target cells. This review details the impact of the mildly acidic milieu of endosomes on the entry of herpesviruses, with particular emphasis on herpes simplex virus 1 (HSV-1). Epithelial cells, the portal of primary HSV-1 infection, support entry via low pH endocytosis mechanisms. Mildly acidic pH triggers reversible conformational changes in the HSV-1 class III fusion protein glycoprotein B (gB). In vitro treatment of herpes simplex virions with a similar pH range inactivates infectivity, likely by prematurely activating the viral entry machinery in the absence of a target membrane. How a given herpesvirus mediates both low pH and pH-independent entry events is a key unresolved question. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

PubMed

Nakajima, Ryota; Takeda, Shizu

2014-01-01

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

Short-interfering RNAs Induce Retinal Degeneration via TLR3 and IRF3

PubMed Central

Kleinman, Mark E; Kaneko, Hiroki; Cho, Won Gil; Dridi, Sami; Fowler, Benjamin J; Blandford, Alexander D; Albuquerque, Romulo JC; Hirano, Yoshio; Terasaki, Hiroko; Kondo, Mineo; Fujita, Takashi; Ambati, Balamurali K; Tarallo, Valeria; Gelfand, Bradley D; Bogdanovich, Sasha; Baffi, Judit Z; Ambati, Jayakrishna

2012-01-01

The discovery of sequence-specific gene silencing by endogenous double-stranded RNAs (dsRNA) has propelled synthetic short-interfering RNAs (siRNAs) to the forefront of targeted pharmaceutical engineering. The first clinical trials utilized 21-nucleotide (nt) siRNAs for the treatment of neovascular age-related macular degeneration (AMD). Surprisingly, these compounds were not formulated for cell permeation, which is required for bona fide RNA interference (RNAi). We showed that these “naked” siRNAs suppress neovascularization in mice not via RNAi but via sequence-independent activation of cell surface Toll-like receptor-3 (TLR3). Here, we demonstrate that noninternalized siRNAs induce retinal degeneration in mice by activating surface TLR3 on retinal pigmented epithelial cells. Cholesterol conjugated siRNAs capable of cell permeation and triggering RNAi also induce the same phenotype. Retinal degeneration was not observed after treatment with siRNAs shorter than 21-nts. Other cytosolic dsRNA sensors are not critical to this response. TLR3 activation triggers caspase-3-mediated apoptotic death of the retinal pigment epithelium (RPE) via nuclear translocation of interferon regulatory factor-3. While this unexpected adverse effect of siRNAs has implications for future clinical trials, these findings also introduce a new preclinical model of geographic atrophy (GA), a late stage of dry AMD that causes blindness in millions worldwide. PMID:21988875

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

PubMed

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

2017-05-02

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

Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

PubMed Central

Ando, Seijitsu; Otani, Hitomi; Yagi, Yasuhiro; Kawai, Kenzo; Araki, Hiromasa; Fukuhara, Shirou; Inagaki, Chiyoko

2007-01-01

Background Proteinase-activated receptors (PARs; PAR1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT) which contributes to the increase in myofibroblast population. Methods EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA) for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells). Results Stimulation of PAR with thrombin (1 U/ml) or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM) for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β). Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR) kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. Conclusion PAR4 stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT) as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation. PMID:17433115

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

PubMed

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

2018-04-01

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

Sequestration of human cytomegalovirus by human renal and mammary epithelial cells

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

Twite, Nicolas; Andrei, Graciela; Kummert, Caroline

2014-07-15

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

Activated ERK1/2 increases CD44 in glomerular parietal epithelial cells leading to matrix expansion

PubMed Central

Roeder, Sebastian S.; Barnes, Taylor J.; Lee, Jonathan S.; Kato, India; Eng, Diana G.; Kaverina, Natalya V.; Sunseri, Maria W.; Daniel, Christoph; Amann, Kerstin; Pippin, Jeffrey W.; Shankland, Stuart J.

2017-01-01

The glycoprotein CD44 is barely detected in normal mouse and human glomeruli, but is increased in glomerular parietal epithelial cells following podocyte injury in focal segmental glomerulosclerosis (FSGS). To determine the biological role and regulation of CD44 in these cells, we employed an in vivo and in vitro approach. Experimental FSGS was induced in CD44 knockout and wildtype mice with a cytotoxic podocyte antibody. Albuminuria, focal and global glomerulosclerosis (periodic acid-Schiff stain) and collagen IV staining were lower in CD44 knockout compared with wild type mice with FSGS. Parietal epithelial cells had lower migration from Bowman’s capsule to the glomerular tuft in CD44 knockout mice with disease compared with wild type mice. In cultured murine parietal epithelial cells, overexpressing CD44 with a retroviral vector encoding CD44 was accompanied by significantly increased collagen IV expression and parietal epithelial cells migration. Because our results showed de novo co-staining for activated ERK1/2 (pERK) in parietal epithelial cells in experimental FSGS, and also in biopsies from patients with FSGS, two in vitro strategies were employed to prove that pERK regulated CD44 levels. First, mouse parietal epithelial cells were infected with a retroviral vector for the upstream kinase MEK-DD to increase pERK, which was accompanied by increased CD44 levels. Second, in CD44 overexpressing parietal epithelial cells, decreasing pERK with U0126 was accompanied by reduced CD44. Finally, parietal epithelial cell migration was higher in cells with increased and reduced in cells with decreased pERK. Thus, pERK is a regulator of CD44 expression and increased CD44 expression leads to a pro-sclerotic and migratory parietal epithelial cells phenotype. PMID:27998643

Progesterone-induced miR-133a inhibits the proliferation of endometrial epithelial cells.

PubMed

Pan, J-L; Yuan, D-Z; Zhao, Y-B; Nie, L; Lei, Y; Liu, M; Long, Y; Zhang, J-H; Blok, L J; Burger, C W; Yue, L-M

2017-03-01

This study aimed to understand the role of miR-133a in progesterone actions, explore the regulative mechanism of the progesterone receptor, and investigate the effects of miR-133a on the progesterone-inhibited proliferation of mouse endometrial epithelial cells. The expression of miR-133a induced by progesterone was detected by quantitative real-time PCR both in vivo and in vitro. Ishikawa subcell lines stably transfected with progesterone receptor subtypes were used to determine the receptor mechanism of progesterone inducing miR-133a. Specific miR-133a mimics or inhibitors were transfected into mouse uteri and primary cultured endometrial epithelial cells to overexpress or downregulate the miR-133a. The roles of miR-133a in the cell cycle and proliferation of endometrial epithelial cells were analysed by flow cytometry and Edu incorporation analysis. The protein levels of cyclinD2 in uterine tissue sections and primary cultured endometrial epithelial cells were determined by immunohistochemistry and Western blot analysis. Progesterone could induce miR-133a expression in a PRB-dependent manner in endometrial epithelial cells. miR-133a inhibited endometrial epithelial cell proliferation by arresting cell cycle at the G 1 -S transition. Moreover, miR-133a acted as an inhibitor in downregulating cyclinD2 in endometrial epithelial cells. We showed for the first time that progesterone-induced miR-133a inhibited the proliferation of endometrial epithelial cells by downregulating cyclinD2. Our research indicated an important mechanism for progesterone inhibiting the proliferation of endometrial epithelial cells by inducing special miRNAs to inhibit positive regulatory proteins in the cell cycle. © 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Epithelial cells as alternative human biomatrices for comet assay.

PubMed

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

2014-01-01

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

Epithelial cells as alternative human biomatrices for comet assay

PubMed Central

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

2014-01-01

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

Induction of innate immunity and its perturbation by influenza viruses.

PubMed

Goraya, Mohsan Ullah; Wang, Song; Munir, Muhammad; Chen, Ji-Long

2015-10-01

Influenza A viruses (IAV) are highly contagious pathogens causing dreadful losses to human and animal, around the globe. IAVs first interact with the host through epithelial cells, and the viral RNA containing a 5'-triphosphate group is thought to be the critical trigger for activation of effective innate immunity via pattern recognition receptors-dependent signaling pathways. These induced immune responses establish the antiviral state of the host for effective suppression of viral replication and enhancing viral clearance. However, IAVs have evolved a variety of mechanisms by which they can invade host cells, circumvent the host immune responses, and use the machineries of host cells to synthesize and transport their own components, which help them to establish a successful infection and replication. In this review, we will highlight the molecular mechanisms of how IAV infection stimulates the host innate immune system and strategies by which IAV evades host responses.

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

PubMed Central

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

2017-01-01

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

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

PubMed

Patil, Sharvari Vijaykumar; Nanduri, Lalitha S Y

2017-11-01

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

A Mechanistic Understanding of Pyroptosis: The Fiery Death Triggered by Invasive Infection.

PubMed

Liu, Xing; Lieberman, Judy

2017-01-01

Immune cells and skin and mucosal epithelial cells recognize invasive microbes and other signs of danger to sound alarms that recruit responder cells and initiate an immediate "innate" immune response. An especially powerful alarm is triggered by cytosolic sensors of invasive infection that assemble into multimolecular complexes, called inflammasomes, that activate the inflammatory caspases, leading to maturation and secretion of proinflammatory cytokines and pyroptosis, an inflammatory death of the infected cell. Work in the past year has defined the molecular basis of pyroptosis. Activated inflammatory caspases cleave Gasdermin D (GSDMD), a cytosolic protein in immune antigen-presenting cells and epithelia. Cleavage separates the autoinhibitory C-terminal fragment from the active N-terminal fragment, which moves to the cell membrane, binds to lipids on the inside of the cell membrane, and oligomerizes to form membrane pores that disrupt cell membrane integrity, causing death and leakage of small molecules, including the proinflammatory cytokines and GSDMD itself. GSDMD also binds to cardiolipin on bacterial membranes and kills the very bacteria that activate the inflammasome. GSDMD belongs to a family of poorly studied gasdermins, expressed in the skin and mucosa, which can also form membrane pores. Spontaneous mutations that disrupt the binding of the N- and C-terminal domains of other gasdermins are associated with alopecia and asthma. Here, we review recent studies that identified the roles of the inflammasome, inflammatory caspases, and GSDMD in pyroptosis and highlight some of the outstanding questions about their roles in innate immunity, control of infection, and sepsis. © 2017 Elsevier Inc. All rights reserved.

Cigarette Smoke Modulates Repair and Innate Immunity following Injury to Airway Epithelial Cells.

PubMed

Amatngalim, Gimano D; Broekman, Winifred; Daniel, Nadia M; van der Vlugt, Luciën E P M; van Schadewijk, Annemarie; Taube, Christian; Hiemstra, Pieter S

2016-01-01

Cigarette smoking is the main risk factor associated with chronic obstructive pulmonary disease (COPD), and contributes to COPD development and progression by causing epithelial injury and inflammation. Whereas it is known that cigarette smoke (CS) may affect the innate immune function of airway epithelial cells and epithelial repair, this has so far not been explored in an integrated design using mucociliary differentiated airway epithelial cells. In this study, we examined the effect of whole CS exposure on wound repair and the innate immune activity of mucociliary differentiated primary bronchial epithelial cells, upon injury induced by disruption of epithelial barrier integrity or by mechanical wounding. Upon mechanical injury CS caused a delayed recovery in the epithelial barrier integrity and wound closure. Furthermore CS enhanced innate immune responses, as demonstrated by increased expression of the antimicrobial protein RNase 7. These differential effects on epithelial repair and innate immunity were both mediated by CS-induced oxidative stress. Overall, our findings demonstrate modulation of wound repair and innate immune responses of injured airway epithelial cells that may contribute to COPD development and progression.

An mDia2/ROCK Signaling Axis Regulates Invasive Egress from Epithelial Ovarian Cancer Spheroids

PubMed Central

Pettee, Krista M.; Dvorak, Kaitlyn M.; Nestor-Kalinoski, Andrea L.; Eisenmann, Kathryn M.

2014-01-01

Multi-cellular spheroids are enriched in ascites of epithelial ovarian cancer (OvCa) patients. They represent an invasive and chemoresistant cellular population fundamental to metastatic dissemination. The molecular mechanisms triggering single cell invasive egress from spheroids remain enigmatic. mDia formins are Rho GTPase effectors that are key regulators of F-actin cytoskeletal dynamics. We hypothesized that mDia2-driven F-actin dynamics promote single cell invasive transitions in clinically relevant three-dimensional (3D) OvCa spheroids. The current study is a dissection of the contribution of the F-actin assembly factor mDia2 formin in invasive transitions and using a clinically relevant ovarian cancer spheroid model. We show that RhoA-directed mDia2 activity is required for tight spheroid organization, and enrichment of mDia2 in the invasive cellular protrusions of collagen-embedded OVCA429 spheroids. Depleting mDia2 in ES-2 spheroids enhanced invasive dissemination of single amoeboid-shaped cells. This contrasts with spheroids treated with control siRNA, where a mesenchymal invasion program predominated. Inhibition of another RhoA effector, ROCK, had no impact on ES-2 spheroid formation but dramatically inhibited spheroid invasion through induction of a highly elongated morphology. Concurrent inhibition of ROCK and mDia2 blocked single cell invasion from ES-2 spheroids more effectively than inhibition of either protein alone, indicating that invasive egress of amoeboid cells from mDia2-depleted spheroids is ROCK-dependent. Our findings indicate that multiple GTPase effectors must be suppressed in order to fully block invasive egress from ovarian cancer spheroids. Furthermore, tightly regulated interplay between ROCK and mDia2 signaling pathways dictates the invasive capacities and the type of invasion program utilized by motile spheroid-derived ovarian cancer cells. As loss of the gene encoding mDia2, DRF3, has been linked to cancer progression and metastasis, our results set the stage for understanding molecular mechanisms involved in mDia2-dependent egress of invasive cells from primary epithelial tumors. PMID:24587343

An mDia2/ROCK signaling axis regulates invasive egress from epithelial ovarian cancer spheroids.

PubMed

Pettee, Krista M; Dvorak, Kaitlyn M; Nestor-Kalinoski, Andrea L; Eisenmann, Kathryn M

2014-01-01

Multi-cellular spheroids are enriched in ascites of epithelial ovarian cancer (OvCa) patients. They represent an invasive and chemoresistant cellular population fundamental to metastatic dissemination. The molecular mechanisms triggering single cell invasive egress from spheroids remain enigmatic. mDia formins are Rho GTPase effectors that are key regulators of F-actin cytoskeletal dynamics. We hypothesized that mDia2-driven F-actin dynamics promote single cell invasive transitions in clinically relevant three-dimensional (3D) OvCa spheroids. The current study is a dissection of the contribution of the F-actin assembly factor mDia2 formin in invasive transitions and using a clinically relevant ovarian cancer spheroid model. We show that RhoA-directed mDia2 activity is required for tight spheroid organization, and enrichment of mDia2 in the invasive cellular protrusions of collagen-embedded OVCA429 spheroids. Depleting mDia2 in ES-2 spheroids enhanced invasive dissemination of single amoeboid-shaped cells. This contrasts with spheroids treated with control siRNA, where a mesenchymal invasion program predominated. Inhibition of another RhoA effector, ROCK, had no impact on ES-2 spheroid formation but dramatically inhibited spheroid invasion through induction of a highly elongated morphology. Concurrent inhibition of ROCK and mDia2 blocked single cell invasion from ES-2 spheroids more effectively than inhibition of either protein alone, indicating that invasive egress of amoeboid cells from mDia2-depleted spheroids is ROCK-dependent. Our findings indicate that multiple GTPase effectors must be suppressed in order to fully block invasive egress from ovarian cancer spheroids. Furthermore, tightly regulated interplay between ROCK and mDia2 signaling pathways dictates the invasive capacities and the type of invasion program utilized by motile spheroid-derived ovarian cancer cells. As loss of the gene encoding mDia2, DRF3, has been linked to cancer progression and metastasis, our results set the stage for understanding molecular mechanisms involved in mDia2-dependent egress of invasive cells from primary epithelial tumors.

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

PubMed Central

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

2011-01-01

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

Precocious development of lectin (Ulex europaeus agglutinin I) receptors in dome epithelium of gut-associated lymphoid tissues.

PubMed

Roy, M J

1987-06-01

Dome epithelium (DE), the tissue covering lymphoid domes of gut-associated lymphoid tissues, was examined in both adult and neonatal rabbit appendix or sacculus rotundus to determine if dome epithelial cells matured earlier than epithelial cells covering adjacent villi. The localization of well-differentiated epithelial cells in rabbit gut-associated lymphoid tissues (GALT) was accomplished histochemically by use of molecular probes: fluorescein isothiocyanate or horseradish peroxidase conjugates of Ulex europaeus agglutinin I (UEA), a lectin specific for terminal L-fucose molecules on certain glycoconjugates. The villus epithelial cells of newborn and 2-, 5-, or 10-day-old rabbits did not bind UEA, but between the twelfth and fifteenth days of postnatal life, UEA receptors were expressed by well-differentiated villus epithelial cells. In contrast to villus epithelium, DE in appendix and sacculus rotundus of neonatal rabbits expressed UEA receptors two days after birth, a feature that distinguished the DE of neonatal GALT for the next two weeks. In adult rabbits, UEA receptors were associated with dome epithelial cells extending from the mouths of glandular crypts to the upper domes; in contrast to the domes, UEA receptors were only present on well-differentiated epithelial cells at the villus tips. Results suggested that in neonatal rabbits most dome epithelial cells developed UEA receptors shortly after birth, reflecting precocious development of DE as compared to villus epithelium. In adult rabbit dome epithelium UEA receptors appeared on dome epithelial cells as they left the glandular crypts, representing accelerated epithelial maturation.

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

PubMed

Sweat JMDunigan, D D; Wright, S D

2001-06-01

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

A Network Biology Approach Identifies Molecular Cross-Talk between Normal Prostate Epithelial and Prostate Carcinoma Cells

PubMed Central

Trevino, Victor; Cassese, Alberto; Nagy, Zsuzsanna; Zhuang, Xiaodong; Herbert, John; Antzack, Philipp; Clarke, Kim; Davies, Nicholas; Rahman, Ayesha; Campbell, Moray J.; Bicknell, Roy; Vannucci, Marina; Falciani, Francesco

2016-01-01

Abstract The advent of functional genomics has enabled the genome-wide characterization of the molecular state of cells and tissues, virtually at every level of biological organization. The difficulty in organizing and mining this unprecedented amount of information has stimulated the development of computational methods designed to infer the underlying structure of regulatory networks from observational data. These important developments had a profound impact in biological sciences since they triggered the development of a novel data-driven investigative approach. In cancer research, this strategy has been particularly successful. It has contributed to the identification of novel biomarkers, to a better characterization of disease heterogeneity and to a more in depth understanding of cancer pathophysiology. However, so far these approaches have not explicitly addressed the challenge of identifying networks representing the interaction of different cell types in a complex tissue. Since these interactions represent an essential part of the biology of both diseased and healthy tissues, it is of paramount importance that this challenge is addressed. Here we report the definition of a network reverse engineering strategy designed to infer directional signals linking adjacent cell types within a complex tissue. The application of this inference strategy to prostate cancer genome-wide expression profiling data validated the approach and revealed that normal epithelial cells exert an anti-tumour activity on prostate carcinoma cells. Moreover, by using a Bayesian hierarchical model integrating genetics and gene expression data and combining this with survival analysis, we show that the expression of putative cell communication genes related to focal adhesion and secretion is affected by epistatic gene copy number variation and it is predictive of patient survival. Ultimately, this study represents a generalizable approach to the challenge of deciphering cell communication networks in a wide spectrum of biological systems. PMID:27124473

A Network Biology Approach Identifies Molecular Cross-Talk between Normal Prostate Epithelial and Prostate Carcinoma Cells.

PubMed

Trevino, Victor; Cassese, Alberto; Nagy, Zsuzsanna; Zhuang, Xiaodong; Herbert, John; Antczak, Philipp; Clarke, Kim; Davies, Nicholas; Rahman, Ayesha; Campbell, Moray J; Guindani, Michele; Bicknell, Roy; Vannucci, Marina; Falciani, Francesco

2016-04-01

The advent of functional genomics has enabled the genome-wide characterization of the molecular state of cells and tissues, virtually at every level of biological organization. The difficulty in organizing and mining this unprecedented amount of information has stimulated the development of computational methods designed to infer the underlying structure of regulatory networks from observational data. These important developments had a profound impact in biological sciences since they triggered the development of a novel data-driven investigative approach. In cancer research, this strategy has been particularly successful. It has contributed to the identification of novel biomarkers, to a better characterization of disease heterogeneity and to a more in depth understanding of cancer pathophysiology. However, so far these approaches have not explicitly addressed the challenge of identifying networks representing the interaction of different cell types in a complex tissue. Since these interactions represent an essential part of the biology of both diseased and healthy tissues, it is of paramount importance that this challenge is addressed. Here we report the definition of a network reverse engineering strategy designed to infer directional signals linking adjacent cell types within a complex tissue. The application of this inference strategy to prostate cancer genome-wide expression profiling data validated the approach and revealed that normal epithelial cells exert an anti-tumour activity on prostate carcinoma cells. Moreover, by using a Bayesian hierarchical model integrating genetics and gene expression data and combining this with survival analysis, we show that the expression of putative cell communication genes related to focal adhesion and secretion is affected by epistatic gene copy number variation and it is predictive of patient survival. Ultimately, this study represents a generalizable approach to the challenge of deciphering cell communication networks in a wide spectrum of biological systems.

Highly pathogenic avian influenza H5N1 virus delays apoptotic responses via activation of STAT3

PubMed Central

Hui, Kenrie P. Y.; Li, Hung Sing; Cheung, Man Chun; Chan, Renee W. Y.; Yuen, Kit M.; Mok, Chris K. P.; Nicholls, John M.; Peiris, J. S. Malik; Chan, Michael C. W.

2016-01-01

Highly pathogenic avian influenza (HPAI) H5N1 virus continues to pose pandemic threat, but there is a lack of understanding of its pathogenesis. We compared the apoptotic responses triggered by HPAI H5N1 and low pathogenic H1N1 viruses using physiologically relevant respiratory epithelial cells. We demonstrated that H5N1 viruses delayed apoptosis in primary human bronchial and alveolar epithelial cells (AECs) compared to H1N1 virus. Both caspase-8 and -9 were activated by H5N1 and H1N1 viruses in AECs, while H5N1 differentially up-regulated TRAIL. H5N1-induced apoptosis was reduced by TRAIL receptor silencing. More importantly, STAT3 knock-down increased apoptosis by H5N1 infection suggesting that H5N1 virus delays apoptosis through activation of STAT3. Taken together, we demonstrate that STAT3 is involved in H5N1-delayed apoptosis compared to H1N1. Since delay in apoptosis prolongs the duration of virus replication and production of pro-inflammatory cytokines and TRAIL from H5N1-infected cells, which contribute to orchestrate cytokine storm and tissue damage, our results suggest that STAT3 may play a previously unsuspected role in H5N1 pathogenesis. PMID:27344974

A Phosphoinositide 3-Kinase (PI3K)-serum- and glucocorticoid-inducible Kinase 1 (SGK1) Pathway Promotes Kv7.1 Channel Surface Expression by Inhibiting Nedd4-2 Protein*

PubMed Central

Andersen, Martin Nybo; Krzystanek, Katarzyna; Petersen, Frederic; Bomholtz, Sofia Hammami; Olesen, Søren-Peter; Abriel, Hugues; Jespersen, Thomas; Rasmussen, Hanne Borger

2013-01-01

Epithelial cell polarization involves several kinase signaling cascades that eventually divide the surface membrane into an apical and a basolateral part. One kinase, which is activated during the polarization process, is phosphoinositide 3-kinase (PI3K). In MDCK cells, the basolateral potassium channel Kv7.1 requires PI3K activity for surface-expression during the polarization process. Here, we demonstrate that Kv7.1 surface expression requires tonic PI3K activity as PI3K inhibition triggers endocytosis of these channels in polarized MDCK. Pharmacological inhibition of SGK1 gave similar results as PI3K inhibition, whereas overexpression of constitutively active SGK1 overruled it, suggesting that SGK1 is the primary downstream target of PI3K in this process. Furthermore, knockdown of the ubiquitin ligase Nedd4-2 overruled PI3K inhibition, whereas a Nedd4-2 interaction-deficient Kv7.1 mutant was resistant to both PI3K and SGK1 inhibition. Altogether, these data suggest that a PI3K-SGK1 pathway stabilizes Kv7.1 surface expression by inhibiting Nedd4-2-dependent endocytosis and thereby demonstrates that Nedd4-2 is a key regulator of Kv7.1 localization and turnover in epithelial cells. PMID:24214981

A phosphoinositide 3-kinase (PI3K)-serum- and glucocorticoid-inducible kinase 1 (SGK1) pathway promotes Kv7.1 channel surface expression by inhibiting Nedd4-2 protein.

PubMed

Andersen, Martin Nybo; Krzystanek, Katarzyna; Petersen, Frederic; Bomholtz, Sofia Hammami; Olesen, Søren-Peter; Abriel, Hugues; Jespersen, Thomas; Rasmussen, Hanne Borger

2013-12-27

Epithelial cell polarization involves several kinase signaling cascades that eventually divide the surface membrane into an apical and a basolateral part. One kinase, which is activated during the polarization process, is phosphoinositide 3-kinase (PI3K). In MDCK cells, the basolateral potassium channel Kv7.1 requires PI3K activity for surface-expression during the polarization process. Here, we demonstrate that Kv7.1 surface expression requires tonic PI3K activity as PI3K inhibition triggers endocytosis of these channels in polarized MDCK. Pharmacological inhibition of SGK1 gave similar results as PI3K inhibition, whereas overexpression of constitutively active SGK1 overruled it, suggesting that SGK1 is the primary downstream target of PI3K in this process. Furthermore, knockdown of the ubiquitin ligase Nedd4-2 overruled PI3K inhibition, whereas a Nedd4-2 interaction-deficient Kv7.1 mutant was resistant to both PI3K and SGK1 inhibition. Altogether, these data suggest that a PI3K-SGK1 pathway stabilizes Kv7.1 surface expression by inhibiting Nedd4-2-dependent endocytosis and thereby demonstrates that Nedd4-2 is a key regulator of Kv7.1 localization and turnover in epithelial cells.

Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals.

PubMed

Tizzano, Marco; Gulbransen, Brian D; Vandenbeuch, Aurelie; Clapp, Tod R; Herman, Jake P; Sibhatu, Hiruy M; Churchill, Mair E A; Silver, Wayne L; Kinnamon, Sue C; Finger, Thomas E

2010-02-16

The upper respiratory tract is continually assaulted with harmful dusts and xenobiotics carried on the incoming airstream. Detection of such irritants by the trigeminal nerve evokes protective reflexes, including sneezing, apnea, and local neurogenic inflammation of the mucosa. Although free intra-epithelial nerve endings can detect certain lipophilic irritants (e.g., mints, ammonia), the epithelium also houses a population of trigeminally innervated solitary chemosensory cells (SCCs) that express T2R bitter taste receptors along with their downstream signaling components. These SCCs have been postulated to enhance the chemoresponsive capabilities of the trigeminal irritant-detection system. Here we show that transduction by the intranasal solitary chemosensory cells is necessary to evoke trigeminally mediated reflex reactions to some irritants including acyl-homoserine lactone bacterial quorum-sensing molecules, which activate the downstream signaling effectors associated with bitter taste transduction. Isolated nasal chemosensory cells respond to the classic bitter ligand denatonium as well as to the bacterial signals by increasing intracellular Ca(2+). Furthermore, these same substances evoke changes in respiration indicative of trigeminal activation. Genetic ablation of either G alpha-gustducin or TrpM5, essential elements of the T2R transduction cascade, eliminates the trigeminal response. Because acyl-homoserine lactones serve as quorum-sensing molecules for gram-negative pathogenic bacteria, detection of these substances by airway chemoreceptors offers a means by which the airway epithelium may trigger an epithelial inflammatory response before the bacteria reach population densities capable of forming destructive biofilms.

Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals

PubMed Central

Tizzano, Marco; Gulbransen, Brian D.; Vandenbeuch, Aurelie; Clapp, Tod R.; Herman, Jake P.; Sibhatu, Hiruy M.; Churchill, Mair E. A.; Silver, Wayne L.; Kinnamon, Sue C.; Finger, Thomas E.

2010-01-01

The upper respiratory tract is continually assaulted with harmful dusts and xenobiotics carried on the incoming airstream. Detection of such irritants by the trigeminal nerve evokes protective reflexes, including sneezing, apnea, and local neurogenic inflammation of the mucosa. Although free intra-epithelial nerve endings can detect certain lipophilic irritants (e.g., mints, ammonia), the epithelium also houses a population of trigeminally innervated solitary chemosensory cells (SCCs) that express T2R bitter taste receptors along with their downstream signaling components. These SCCs have been postulated to enhance the chemoresponsive capabilities of the trigeminal irritant-detection system. Here we show that transduction by the intranasal solitary chemosensory cells is necessary to evoke trigeminally mediated reflex reactions to some irritants including acyl–homoserine lactone bacterial quorum-sensing molecules, which activate the downstream signaling effectors associated with bitter taste transduction. Isolated nasal chemosensory cells respond to the classic bitter ligand denatonium as well as to the bacterial signals by increasing intracellular Ca2+. Furthermore, these same substances evoke changes in respiration indicative of trigeminal activation. Genetic ablation of either Gα-gustducin or TrpM5, essential elements of the T2R transduction cascade, eliminates the trigeminal response. Because acyl–homoserine lactones serve as quorum-sensing molecules for Gram-negative pathogenic bacteria, detection of these substances by airway chemoreceptors offers a means by which the airway epithelium may trigger an epithelial inflammatory response before the bacteria reach population densities capable of forming destructive biofilms. PMID:20133764

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

PubMed

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

2015-09-01

To explore the biocompatibility of the poly-lactide-co-glycolide (PLGA)/collagen type I scaffold with rat vaginal epithelial cells, and the feasibility of using PLGA/collagen type I as scaffold to reconstruct vagina by the tissue engineering. PLGA/collagen type I scaffold was prepared with PLGA covered polylysine and collagen type I. The vaginal epithelial cells of Sprague Dawley rat of 10-12 weeks old were cultured by enzyme digestion method. The vaginal epithelial cells of passage 2 were cultured in the leaching liquor of scaffold for 48 hours to detect its cytotoxicity by MTT. The vaginal epithelial cells were inoculated on the PLGA/collagen type I scaffold (experimental group) and PLGA scaffold (control group) to calculate the cell adhesion rate. Epithelial cells-scaffold complexes were implanted subcutaneously on the rat back. At 2, 4, and 8 weeks after implantation, the epithelial cells-scaffold complexes were harvested to observe the cell growth by HE staining and immunohistochemical analysis. The epithelial cells-scaffold complexes were transplanted to reconstruct vagina in 6 rats with vaginal defect. After 3 and 6 months, the vaginal length was measured and the appearance was observed. The neovagina tissues were harvested for histological evaluation after 6 months. The epithelial cells grew and proliferated well in the leaching liquor of PLGA/collagen type I scaffold, and the cytotoxicity was at grade 1. The cell adhesion rate on the PLGA/collagen type I scaffold was 71.8%±9.2%, which significantly higher than that on the PLGA scaffold (63.4%±5.7%) (t=2.195, P=0.005). The epithelial cells could grow and adhere to the PLGA/collagen type I scaffolds. At 2 weeks after implanted subcutaneously, the epithelial cells grew and proliferated in the pores of scaffolds, and the fibroblasts were observed. At 4 weeks, 1-3 layers epithelium formed on the surface of scaffold. At 8 weeks, the epithelial cells increased and arranged regularly, which formed the membrane-like layer on the scaffold. The keratin expression of the epithelium was positive. At 3 months after transplantation in situ, the vaginal mucosa showed pink and lustrous epithelialization, and the majority of scaffold degraded. After 6 months, the neovagina length was 1.2 cm, without obvious stenosis; the vaginal mucosa had similar appearance and epithelial layer to normal vagina, but it had less duplicature; there were nail-like processes in the basal layer, but the number was less than that of normal vagina. The immunohistochemistry staining for keratin was positive. The PLGA/collagen type I scaffolds have good cytocompatibility with the epithelial cells, and can be used as the biodegradable polymer scaffold of the vaginal tissue engineering.

Hypoxia induced EMT: A review on the mechanism of tumor progression and metastasis in OSCC.

PubMed

Joseph, Joel P; Harishankar, M K; Pillai, Aruthra Arumugam; Devi, Arikketh

2018-05-01

Hypoxia, a condition of low oxygen tension in tissues, has emerged as a crucial factor in tumor pathophysiology. Hypoxic microenvironment gives rise to altered cellular metabolism and triggers varied molecular responses. These responses promote tumor progression and confer radiation resistance and chemo resistance to tumors. The predominant molecules that are associated with hypoxia research are the hypoxia inducible factors (HIFs). HIFs are known to regulate a large group of genes that are involved in cell survival, proliferation, motility, metabolism, pH regulation, extracellular matrix function, inflammatory cell recruitment and angiogenesis by inducing the expression of their downstream target genes. The process of epithelial to mesenchymal transition (EMT) has been associated with metastasis in cancer. Reports also suggest that hypoxia triggers EMT in several types of cancer including breast cancer, prostate cancer and oral cancer. Oral cancer is a predominant cancer in Central and South East Asia. However, in the recent times, the incidence rates of oral cancer have been increasing in Northern and Eastern Europe as well. This review articulates the role of hypoxia and the associated factors like HIFs in inducing EMT in oral cancer (OSCC). Copyright © 2018 Elsevier Ltd. All rights reserved.

TASK-3 Downregulation Triggers Cellular Senescence and Growth Inhibition in Breast Cancer Cell Lines.

PubMed

Zúñiga, Rafael; Valenzuela, Claudio; Concha, Guierdy; Brown, Nelson; Zúñiga, Leandro

2018-03-29

TASK-3 potassium channels are believed to promote proliferation and survival of cancer cells, in part, by augmenting their resistance to both hypoxia and serum deprivation. While overexpression of TASK-3 is frequently observed in cancers, the understanding of its role and regulation during tumorigenesis remains incomplete. Here, we evaluated the effect of reducing the expression of TASK-3 in MDA-MB-231 and MCF-10F human mammary epithelial cell lines through small hairpin RNA (shRNA)-mediated knockdown. Our results show that knocking down TASK-3 in fully transformed MDA-MB-231 cells reduces proliferation, which was accompanied by an induction of cellular senescence and cell cycle arrest, with an upregulation of cyclin-dependent kinase (CDK) inhibitors p21 and p27. In non-tumorigenic MCF-10F cells, however, TASK-3 downregulation did not lead to senescence induction, although cell proliferation was impaired and an upregulation of CDK inhibitors was also evident. Our observations implicate TASK-3 as a critical factor in cell cycle progression and corroborate its potential as a therapeutic target in breast cancer treatment.

Natural Killer Cells Control Tumor Growth by Sensing a Growth Factor.

PubMed

Barrow, Alexander D; Edeling, Melissa A; Trifonov, Vladimir; Luo, Jingqin; Goyal, Piyush; Bohl, Benjamin; Bando, Jennifer K; Kim, Albert H; Walker, John; Andahazy, Mary; Bugatti, Mattia; Melocchi, Laura; Vermi, William; Fremont, Daved H; Cox, Sarah; Cella, Marina; Schmedt, Christian; Colonna, Marco

2018-01-25

Many tumors produce platelet-derived growth factor (PDGF)-DD, which promotes cellular proliferation, epithelial-mesenchymal transition, stromal reaction, and angiogenesis through autocrine and paracrine PDGFRβ signaling. By screening a secretome library, we found that the human immunoreceptor NKp44, encoded by NCR2 and expressed on natural killer (NK) cells and innate lymphoid cells, recognizes PDGF-DD. PDGF-DD engagement of NKp44 triggered NK cell secretion of interferon gamma (IFN)-γ and tumor necrosis factor alpha (TNF-α) that induced tumor cell growth arrest. A distinctive transcriptional signature of PDGF-DD-induced cytokines and the downregulation of tumor cell-cycle genes correlated with NCR2 expression and greater survival in glioblastoma. NKp44 expression in mouse NK cells controlled the dissemination of tumors expressing PDGF-DD more effectively than control mice, an effect enhanced by blockade of the inhibitory receptor CD96 or CpG-oligonucleotide treatment. Thus, while cancer cell production of PDGF-DD supports tumor growth and stromal reaction, it concomitantly activates innate immune responses to tumor expansion. Copyright © 2017 Elsevier Inc. All rights reserved.

TASK-3 Downregulation Triggers Cellular Senescence and Growth Inhibition in Breast Cancer Cell Lines

PubMed Central

Zúñiga, Rafael; Valenzuela, Claudio; Concha, Guierdy; Brown, Nelson; Zúñiga, Leandro

2018-01-01

TASK-3 potassium channels are believed to promote proliferation and survival of cancer cells, in part, by augmenting their resistance to both hypoxia and serum deprivation. While overexpression of TASK-3 is frequently observed in cancers, the understanding of its role and regulation during tumorigenesis remains incomplete. Here, we evaluated the effect of reducing the expression of TASK-3 in MDA-MB-231 and MCF-10F human mammary epithelial cell lines through small hairpin RNA (shRNA)-mediated knockdown. Our results show that knocking down TASK-3 in fully transformed MDA-MB-231 cells reduces proliferation, which was accompanied by an induction of cellular senescence and cell cycle arrest, with an upregulation of cyclin-dependent kinase (CDK) inhibitors p21 and p27. In non-tumorigenic MCF-10F cells, however, TASK-3 downregulation did not lead to senescence induction, although cell proliferation was impaired and an upregulation of CDK inhibitors was also evident. Our observations implicate TASK-3 as a critical factor in cell cycle progression and corroborate its potential as a therapeutic target in breast cancer treatment. PMID:29596383

Silk Film Topography Directs Collective Epithelial Cell Migration

PubMed Central

Rosenblatt, Mark I.

2012-01-01

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

Physiology and pathophysiology of apoptosis in epithelial cells of the liver, pancreas, and intestine.

PubMed

Jones, B A; Gores, G J

1997-12-01

Cell death of gastrointestinal epithelial cells occurs by a process referred to as apoptosis. In this review, we succinctly define apoptosis and summarize the role of apoptosis in the physiology and pathophysiology of epithelial cells in the liver, pancreas, and small and large intestine. The physiological mediators regulating apoptosis in gastrointestinal epithelial cells, when known, are discussed. Selected pathophysiological consequences of excessive apoptosis and inhibition of apoptosis are used to illustrate the significance of apoptosis in disease processes. These examples demonstrate that excessive apoptosis may result in epithelial cell atrophy, injury, and dysfunction, whereas inhibition of apoptosis results in hyperplasia and promotes malignant transformation. The specific cellular mechanisms responsible for dysregulation of epithelial cell apoptosis during pathophysiological disturbances are emphasized. Potential future areas of physiological research regarding apoptosis in gastrointestinal epithelia are highlighted when appropriate.

CCL20, (gamma)(delta) T cells, and IL-22 in corneal epithelial healing

USDA-ARS?s Scientific Manuscript database

After corneal epithelial abrasion, leukocytes and platelets rapidly enter the corneal stroma, and CCR6 (+) IL-17(+) gamma delta T cells migrate into the epithelium. Gamma delta T-cell-deficient (TCRd(-/-)) mice have significantly reduced inflammation and epithelial wound healing. Epithelial CCL20 mR...

Niches for the Long-Term Maintenance of Tissue-Resident Memory T Cells

PubMed Central

Takamura, Shiki

2018-01-01

Tissue-resident memory T cells (TRM cells) are a population of immune cells that reside in the lymphoid and non-lymphoid organs without recirculation through the blood. These important cells occupy and utilize unique anatomical and physiological niches that are distinct from those for other memory T cell populations, such as central memory T cells in the secondary lymphoid organs and effector memory T cells that circulate through the tissues. CD8+ TRM cells typically localize in the epithelial layers of barrier tissues where they are optimally positioned to act as sentinels to trigger antigen-specific protection against reinfection. CD4+ TRM cells typically localize below the epithelial layers, such as below the basement membrane, and cluster in lymphoid structures designed to optimize interactions with antigen-presenting cells upon reinfection. A key feature of TRM populations is their ability to be maintained in barrier tissues for prolonged periods of time. For example, skin CD8+ TRM cells displace epidermal niches originally occupied by γδ T cells, thereby enabling their stable persistence for years. It is also clear that the long-term maintenance of TRM cells in different microenvironments is dependent on multiple tissue-specific survival cues, although the specific details are poorly understood. However, not all TRM persist over the long term. Recently, we identified a new spatial niche for the maintenance of CD8+ TRM cells in the lung, which is created at the site of tissue regeneration after injury [termed repair-associated memory depots (RAMD)]. The short-lived nature of RAMD potentially explains the short lifespans of CD8+ TRM cells in this particular tissue. Clearly, a better understanding of the niche-dependent maintenance of TRM cells will be important for the development of vaccines designed to promote barrier immunity. In this review, we discuss recent advances in our understanding of the properties and nature of tissue-specific niches that maintain TRM cells in different tissues. PMID:29904388

Polarity, cell division, and out-of-equilibrium dynamics control the growth of epithelial structures

PubMed Central

Cerruti, Benedetta; Puliafito, Alberto; Shewan, Annette M.; Yu, Wei; Combes, Alexander N.; Little, Melissa H.; Chianale, Federica; Primo, Luca; Serini, Guido; Mostov, Keith E.; Celani, Antonio

2013-01-01

The growth of a well-formed epithelial structure is governed by mechanical constraints, cellular apico-basal polarity, and spatially controlled cell division. Here we compared the predictions of a mathematical model of epithelial growth with the morphological analysis of 3D epithelial structures. In both in vitro cyst models and in developing epithelial structures in vivo, epithelial growth could take place close to or far from mechanical equilibrium, and was determined by the hierarchy of time-scales of cell division, cell–cell rearrangements, and lumen dynamics. Equilibrium properties could be inferred by the analysis of cell–cell contact topologies, and the nonequilibrium phenotype was altered by inhibiting ROCK activity. The occurrence of an aberrant multilumen phenotype was linked to fast nonequilibrium growth, even when geometric control of cell division was correctly enforced. We predicted and verified experimentally that slowing down cell division partially rescued a multilumen phenotype induced by altered polarity. These results improve our understanding of the development of epithelial organs and, ultimately, of carcinogenesis. PMID:24145168

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

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

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

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

A novel method for isolation of epithelial cells from ovine esophagus for tissue engineering.

PubMed

Macheiner, Tanja; Kuess, Anna; Dye, Julian; Saxena, Amulya K

2014-01-01

The yield of a critical number of basal epithelial cells with high mitotic rates from native tissue is a challenge in the field of tissue engineering. There are many protocols that use enzymatic methods for isolation of epithelial cells with unsatisfactory results for tissue engineering. This study aimed to develop a protocol for isolating a sufficient number of epithelial cells with a high Proliferating Index from ovine esophagus for tissue engineering applications. Esophageal mucosa was pretreated with dispase-collagenase solution and plated on collagen-coated culture dishes. Distinction of the various types of epithelial cells and developmental stages was done with specific primary antibodies to Cytokeratins and to Proliferating Cell Nuclear Antigen (PCNA). Up to approximately 8100 epithelial cells/mm2 of mucosa tissue were found after one week of migration. Cytokeratin 14 (CK 14) was positive identified in cells even after 83 days. At the same time the Proliferating Index was 71%. Our protocol for isolation of basal epithelial cells was successful to yield sufficient numbers of cells predominantly with proliferative character and without noteworthy negative enzymatic affection. The results at this study offer the possibility of generation critical cell numbers for tissue engineering applications.

The Essential Role of Double-Stranded RNA-Dependent Antiviral Signaling in the Degradation of Nonself Single-Stranded RNA in Nonimmune Cells.

PubMed

Kimura, Sayaka; Matsumiya, Tomoh; Shiba, Yuko; Nakanishi, Michi; Hayakari, Ryo; Kawaguchi, Shogo; Yoshida, Hidemi; Imaizumi, Tadaatsu

2018-06-20

The recognition of nonself dsRNA by retinoic acid-inducible gene-I (RIG-I) leads to the engagement of RIG-I-like receptor signaling. In addition, nonself dsRNA triggers a robust latent RNase (RNase L) activation and leads to the degradation of ribosomal structures and cell death. In contrast, nonself ssRNA is known to be recognized by TLR 7/8 in immune cells such as plasmacytoid dendritic cells and B cells, but little is known regarding the involvement of nonself ssRNA in antiviral signaling in nonimmune cells, including epithelial cells. Moreover, the fate of intracellular nonself ssRNA remains unknown. To address this issue, we developed a quantitative RT-PCR-based approach that monitors the kinetics of nonself ssRNA cleavage following the transfection of HeLa human cervical carcinoma cells, using model nonself ssRNA. We discovered that the degradation of ssRNA is independent of RIG-I and type I IFN signaling because ssRNA did not trigger RIG-I-mediated antiviral signaling. We also found that the kinetics of self (5'-capped) and nonself ssRNA decay were unaltered, suggesting that nonself ssRNA is not recognized by nonimmune cells. We further demonstrated that the cleavage of nonself ssRNA is accelerated when nonself dsRNA is also introduced into cells. In addition, the cleavage of nonself ssRNA is completely abolished by knockdown of RNase L. Overall, our data demonstrate the important role of dsRNA-RNase L in nonself ssRNA degradation and may partly explain the positive regulation of the antiviral responses in nonimmune cells. Copyright © 2018 by The American Association of Immunologists, Inc.

Evaluation of ABCG2 and p63 expression in canine cornea and cultivated corneal epithelial cells.

PubMed

Morita, Maresuke; Fujita, Naoki; Takahashi, Ayaka; Nam, Eun Ryel; Yui, Sho; Chung, Cheng Shu; Kawahara, Naoya; Lin, Hsing Yi; Tsuzuki, Keiko; Nakagawa, Takayuki; Nishimura, Ryohei

2015-01-01

To examine the expressions of ABCG2 and p63 in canine corneal epithelia and to evaluate their significance in corneal regeneration. Canine corneal and limbal epithelial cells were obtained from five healthy beagle dogs. We analyzed the morphological properties of cultivated limbal and corneal epithelial cells. We compared the expressions of ABCG2 and p63 in the limbus and central cornea by immunohistochemistry and real-time quantitative PCR. We analyzed the expression of these markers in cultivated cells by immunocytochemistry and real-time quantitative PCR. The limbal epithelial cells were smaller and proliferated more rapidly than the corneal epithelial cells in primary cultures. The corneal cells failed to be subcultured, whereas the limbal cells could be subcultured with increasing cell size. ABCG2 was localized in the basal layer of the limbal epithelium, and p63 was widely detected in the entire corneal epithelia. ABCG2 expression was significantly higher, and p63 was slightly higher in the limbus compared with the central cornea. ABCG2 was detected only in limbal cells in primary culture, not in corneal cells or passaged limbal cells. p63 was detected in both limbal and corneal cells and decreased gradually in the limbal cells with the cell passages. ABCG2 was localized in canine limbal epithelial cells, and p63 was widely expressed in canine corneal epithelia. ABCG2 and p63 could prove to be useful markers in dogs for putative corneal epithelial stem cells and for corneal epithelial cell proliferation, respectively. © 2014 American College of Veterinary Ophthalmologists.

Diagnosis of colorectal cancer using Raman spectroscopy of laser-trapped single living epithelial cells

NASA Astrophysics Data System (ADS)

Chen, Kun; Qin, Yejun; Zheng, Feng; Sun, Menghong; Shi, Daren

2006-07-01

A single-cell diagnostic technique for epithelial cancers is developed by utilizing laser trapping and Raman spectroscopy to differentiate cancerous and normal epithelial cells. Single-cell suspensions were prepared from surgically removed human colorectal tissues following standard primary culture protocols and examined in a near-infrared laser-trapping Raman spectroscopy system, where living epithelial cells were investigated one by one. A diagnostic model was built on the spectral data obtained from 8 patients and validated by the data from 2 new patients. Our technique has potential applications from epithelial cancer diagnosis to the study of cell dynamics of carcinogenesis.

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

PubMed

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

2014-12-01

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

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

PubMed

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

2017-08-01

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

Comparative Iron Oxide Nanoparticle Cellular Dosimetry and Response in Mice by the Inhalation and Liquid Cell Culture Exposure Routes

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

Teeguarden, Justin G.; Mikheev, Vladimir B.; Minard, Kevin R.

testing the rapidly growing number of nanomaterials requires large scale use of in vitro systems under the presumption that these systems are sufficiently predictive or descriptive of responses in in vivo systems for effective use in hazard ranking. We hypothesized that improved relationships between in vitro and in vivo models of experimental toxicology for nanomaterials would result from placing response data in vitro and in vivo on the same dose scale, the amount of material associated with cells (target cell dose). Methods: Balb/c mice were exposed nose-only to an aerosol of 12.8 nm (68.6 nm CMD, 19.9 mg/m3, 4 hours)more » super paramagnetic iron oxide particles, target cell doses were calculated and biomarkers of response anchored with histological evidence were identified by global transcriptomics. Representative murine epithelial and macrophage cell types were exposed in vitro to the same material in liquid suspension for four hours and levels nanoparticle regulated cytokine transcripts identified in vivo were quantified as a function of measured nanoparticle cellular dose. Results. Target tissue doses of 0.009-0.4 μg SPIO/cm2 lung led to an inflammatory response in the alveolar region characterized by interstitial inflammation and macrophage infiltration. In vitro, higher target tissue doses of ~1.2-4 μg SPIO/ cm2 of cells were required to induce transcriptional regulation of markers of inflammation, CXCL2 CCL3, in C10 lung epithelial cells. Estimated in vivo macrophage SPIO nanoparticle doses ranged from 1-100 pg/cell, and induction of inflammatory markers was observed in vitro in macrophages at doses of 8-35 pg/cell. Conclusions: Application of target tissue dosimetry revealed good correspondence between target cell doses triggering inflammatory processes in vitro and in vivo in the alveolar macrophage population, but not in the epithelial cells of the alveolar region. These findings demonstrate the potential for target tissue dosimetry to enable the more quantitative comparison of in vitro and in vivo systems advance their use for hazard assessment and extrapolation to humans. The mildly inflammogentic cellular doses experienced by mice were similar those calculated for humans exposed to the same at the existing permissible exposure limit of 10 mg/m3 iron oxide (as Fe).« less

Chlamydia trachomatis Is Resistant to Inclusion Ubiquitination and Associated Host Defense in Gamma Interferon-Primed Human Epithelial Cells.

PubMed

Haldar, Arun K; Piro, Anthony S; Finethy, Ryan; Espenschied, Scott T; Brown, Hannah E; Giebel, Amanda M; Frickel, Eva-Maria; Nelson, David E; Coers, Jörn

2016-12-13

The cytokine gamma interferon (IFN-γ) induces cell-autonomous immunity to combat infections with intracellular pathogens, such as the bacterium Chlamydia trachomatis The present study demonstrates that IFN-γ-primed human cells ubiquitinate and eliminate intracellular Chlamydia-containing vacuoles, so-called inclusions. We previously described how IFN-γ-inducible immunity-related GTPases (IRGs) employ ubiquitin systems to mark inclusions for destruction in mouse cells and, furthermore, showed that the rodent pathogen Chlamydia muridarum blocks ubiquitination of its inclusions by interfering with mouse IRG function. Here, we report that ubiquitination of inclusions in human cells is independent of IRG and thus distinct from the murine pathway. We show that C. muridarum is susceptible to inclusion ubiquitination in human cells, while the closely related human pathogen C. trachomatis is resistant. C. muridarum, but not C. trachomatis, inclusions attract several markers of cell-autonomous immunity, including the ubiquitin-binding protein p62, the ubiquitin-like protein LC3, and guanylate-binding protein 1. Consequently, we find that IFN-γ priming of human epithelial cells triggers the elimination of C. muridarum, but not C. trachomatis, inclusions. This newly described defense pathway is independent of indole-2,3-dioxygenase, a known IFN-γ-inducible anti-Chlamydia resistance factor. Collectively, our observations indicate that C. trachomatis evolved mechanisms to avoid a human-specific, ubiquitin-mediated response as part of its unique adaptation to its human host. Chlamydia trachomatis is the leading cause of sexually transmitted bacterial infections and responsible for significant morbidity, including pelvic inflammatory disease, infertility, and ectopic pregnancies in women. As an obligate intracellular pathogen, C. trachomatis is in perpetual conflict with cell-intrinsic defense programs executed by its human host. Our study defines a novel anti-Chlamydia host resistance pathway active in human epithelial cells. This defense program promotes the deposition of the small antimicrobial protein ubiquitin on vacuoles containing Chlamydia We show that this ubiquitin-based resistance pathway of human cells is highly effective against a Chlamydia species adapted to rodents but ineffective against human-adapted C. trachomatis This observation indicates that C. trachomatis evolved strategies to avoid entrapment within ubiquitin-labeled vacuoles as part of its adaptation to the human innate immune system. Copyright © 2016 Haldar et al.

Low- and high-LET radiation drives clonal expansion of lung progenitor cells in vivo

PubMed Central

Farin, Alicia M.; Manzo, Nicholas D.; Kirsch, David G.; Stripp, Barry R.

2015-01-01

Abundant populations of epithelial progenitor cells maintain the epithelium along the proximal-to-distal axis of the airway. Exposure of lung tissue to ionizing radiation leads to tissue remodeling and potential cancer initiation or progression. However, little is known about the effects of ionizing radiation on airway epithelial progenitor cells. We hypothesized that ionizing radiation exposure will alter the behavior of airway epithelial progenitor cells in a radiation dose- and quality-dependent manner. To address this hypothesis, we cultured primary airway epithelial cells isolated from mice exposed to various doses of 320 kVp X-ray or 600 MeV/nucleon 56Fe ions in a 3D epithelial-fibroblast co-culture system. Colony-forming efficiency of the airway epithelial progenitor cells was assessed at culture day 14. In vivo clonogenic and proliferative potentials of airway epithelial progenitor cells were measured after exposure to ionizing radiation by lineage tracing and IdU incorporation. Exposure to both X-rays and 56Fe resulted in a dose dependent decrease in the ability of epithelial progenitors to form colonies in vitro. In vivo evidence for increased clonogenic expansion of epithelial progenitors was observed after exposure to both X-rays and 56Fe. Interestingly, we found no significant increase in the epithelial proliferative index, indicating that ionizing radiation does not promote increased turnover of the airway epithelium. Therefore, we propose a model in which radiation induces a dose-dependent decrease in the pool of available progenitor cells, leaving fewer progenitors able to maintain the airway long-term. This work provides novel insights into the effects of ionizing radiation exposure on airway epithelial progenitor cell behavior. PMID:25564721

Comparison of culture media for ex vivo cultivation of limbal epithelial progenitor cells

PubMed Central

Loureiro, Renata Ruoco; Cristovam, Priscila Cardoso; Martins, Caio Marques; Covre, Joyce Luciana; Sobrinho, Juliana Aparecida; Ricardo, José Reinaldo da Silva; Hazarbassanov, Rossen Myhailov; Höfling-Lima, Ana Luisa; Belfort, Rubens; Nishi, Mauro

2013-01-01

Purpose To compare the effectiveness of three culture media for growth, proliferation, differentiation, and viability of ex vivo cultured limbal epithelial progenitor cells. Methods Limbal epithelial progenitor cell cultures were established from ten human corneal rims and grew on plastic wells in three culture media: supplemental hormonal epithelial medium (SHEM), keratinocyte serum-free medium (KSFM), and Epilife. The performance of culturing limbal epithelial progenitor cells in each medium was evaluated according to the following parameters: growth area of epithelial migration; immunocytochemistry for adenosine 5′-triphosphate-binding cassette member 2 (ABCG2), p63, Ki67, cytokeratin 3 (CK3), and vimentin (VMT) and real-time reverse transcription polymerase chain reaction (RT–PCR) for CK3, ABCG2, and p63, and cell viability using Hoechst staining. Results Limbal epithelial progenitor cells cultivated in SHEM showed a tendency to faster migration, compared to KSFM and Epilife. Immunocytochemical analysis showed that proliferated cells in the SHEM had lower expression for markers related to progenitor epithelial cells (ABCG2) and putative progenitor cells (p63), and a higher percentage of positive cells for differentiated epithelium (CK3) when compared to KSFM and Epilife. In PCR analysis, ABCG2 expression was statistically higher for Epilife compared to SHEM. Expression of p63 was statistically higher for Epilife compared to SHEM and KSFM. However, CK3 expression was statistically lower for KSFM compared to SHEM. Conclusions Based on our findings, we concluded that cells cultured in KSFM and Epilife media presented a higher percentage of limbal epithelial progenitor cells, compared to SHEM. PMID:23378720

Bovine TLR2 and TLR4 mediate Cryptosporidium parvum recognition in bovine intestinal epithelial cells.

PubMed

Yang, Zhengtao; Fu, Yunhe; Gong, Pengtao; Zheng, Jingtong; Liu, Li; Yu, Yuqiang; Li, Jianhua; Li, He; Yang, Ju; Zhang, Xichen

2015-08-01

Cryptosporidium parvum (C. parvum) is an intestinal parasite that causes diarrhea in neonatal calves. It results in significant morbidity of neonatal calves and economic losses for producers worldwide. Innate resistance against C. parvum is thought to depend on engagement of pattern recognition receptors. However, the role of innate responses to C. parvum has not been elucidated in bovine. The aim of this study was to evaluate the role of TLRs in host-cell responses during C. parvum infection of cultured bovine intestinal epithelial cells. The expressions of TLRs in bovine intestinal epithelial cells were detected by qRT-PCR. To determine which, if any, TLRs may play a role in the response of bovine intestinal epithelial cells to C. parvum, the cells were stimulated with C. parvum and the expression of TLRs were tested by qRT-PCR. The expression of NF-κB was detected by western blotting. Further analyses were carried out in bovine TLRs transfected HEK293 cells and by TLRs-DN transfected bovine intestinal epithelial cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs. The expression of TLR2 and TLR4 were up-regulated when bovine intestinal epithelial cells were treated with C. parvum. Meanwhile, C. parvum induced IL-8 production in TLR2 or TLR4/MD-2 transfected HEK293 cells. Moreover, C. parvum induced NF-κB activation and cytokine expression in bovine intestinal epithelial cells. The induction of NF-κB activation and cytokine expression by C. parvum were reduced in TLR2-DN and TLR4-DN transfected cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs, and bovine intestinal epithelial cells recognized and responded to C. parvum via TLR2 and TLR4. Copyright © 2015 Elsevier Ltd. All rights reserved.

Gene-expression profiles of epithelial cells treated with EMD in vitro: analysis using complementary DNA arrays.

PubMed

Kapferer, I; Schmidt, S; Gstir, R; Durstberger, G; Huber, L A; Vietor, I

2011-02-01

During surgical periodontal treatment, EMD is topically applied in order to facilitate regeneration of the periodontal ligament, acellular cementum and alveolar bone. Suppresion of epithelial down-growth is essential for successful periodontal regeneration; however, the underlying mechanisms of how EMD influences epithelial wound healing are poorly understood. In the present study, the effects of EMD on gene-expression profiling in an epithelial cell line (HSC-2) model were investigated. Gene-expression modifications, determined using a comparative genome-wide expression-profiling strategy, were independently validated by quantitative real-time RT-PCR. Additionally, cell cycle, cell growth and in vitro wound-healing assays were conducted. A set of 43 EMD-regulated genes was defined, which may be responsible for the reduced epithelial down-growth upon EMD application. Gene ontology analysis revealed genes that could be attributed to pathways of locomotion, developmental processes and associated processes such as regulation of cell size and cell growth. Additionally, eight regulated genes have previously been reported to take part in the process of epithelial-to-mesenchymal transition. Several independent experimental assays revealed significant inhibition of cell migration, growth and cell cycle by EMD. The set of EMD-regulated genes identified in this study offers the opportunity to clarify mechanisms underlying the effects of EMD on epithelial cells. Reduced epithelial repopulation of the dental root upon periodontal surgery may be the consequence of reduced migration and cell growth, as well as epithelial-to-mesenchymal transition. © 2010 John Wiley & Sons A/S.

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

PubMed Central

Gao, Yang; Li, Shu; Li, Qinglei

2014-01-01

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

The host immune response to Clostridium difficile infection

PubMed Central

2013-01-01

Clostridium difficile infection (CDI) is the most common infectious cause of healthcare-acquired diarrhoea. Outcomes of C. difficile colonization are varied, from asymptomatic carriage to fulminant colitis and death, due in part to the interplay between the pathogenic virulence factors of the bacterium and the counteractive immune responses of the host. Secreted toxins A and B are the major virulence factors of C. difficile and induce a profound inflammatory response by intoxicating intestinal epithelial cells causing proinflammatory cytokine release. Host cell necrosis, vascular permeability and neutrophil infiltration lead to an elevated white cell count, profuse diarrhoea and in severe cases, dehydration, hypoalbuminaemia and toxic megacolon. Other bacterial virulence factors, including surface layer proteins and flagella proteins, are detected by host cell surface signal molecules that trigger downstream cell-mediated immune pathways. Human studies have identified a role for serum and faecal immunoglobulin levels in protection from disease, but the recent development of a mouse model of CDI has enabled studies into the precise molecular interactions that trigger the immune response during infection. Key effector molecules have been identified that can drive towards a protective anti-inflammatory response or a damaging proinflammatory response. The limitations of current antimicrobial therapies for CDI have led to the development of both active and passive immunotherapies, none of which have, as yet been formally approved for CDI. However, recent advances in our understanding of the molecular basis of host immune protection against CDI may provide an exciting opportunity for novel therapeutic developments in the future. PMID:25165542

Lacritin and Other New Proteins of the Lacrimal Functional Unit

PubMed Central

McKown, Robert L.; Wang, Ningning; Raab, Ronald W.; Karnati, Roy; Zhang, Yinghui; Williams, Patricia B.; Laurie, Gordon W.

2009-01-01

The lacrimal functional unit (LFU) is defined by the 2007 International Dry Eye WorkShop as ‘an integrated system comprising the lacrimal glands, ocular surface (cornea, conjunctiva and meibomian glands) and lids, and the sensory and motor nerves that connect them’. The LFU maintains a healthy ocular surface primarily through a properly functioning tear film that provides protection, lubrication, and an environment for corneal epithelial cell renewal. LFU cells express thousands of proteins. Over two hundred new LFU proteins have been discovered in the last decade. Lacritin is a new LFU-specific growth factor in human tears that flows through ducts to target corneal epithelial cells on the ocular surface. When applied topically in rabbits, lacritin appears to increase the volume of basal tear secretion. Lacritin is one of only a handful of tear proteins preliminarily reported to be downregulated in blepharitis and in two dry eye syndromes. Computational analysis predicts an ordered C-terminal domain that binds the corneal epithelial cell surface proteoglycan syndecan-1 (SDC1) and is required for lacritin’s low nanomolar mitogenic activity. The lacritin binding site on the N-terminus of SDC1 is exposed by heparanase. Heparanase is constitutively expressed by the corneal epithelium and appears to be a normal constituent of tears. Binding triggers rapid signaling to downstream NFAT and mTOR. A wealth of other new proteins, originally designated as hypothetical when first identified by genomic sequencing, are expressed by the human LFU including: ALS2CL, ARHGEF19, KIAA1109, PLXNA1, POLG, WIPI1 and ZMIZ2. Their demonstrated or implied roles in human genetic disease or basic cellular functions are fuel for new investigation. Addressing topical areas in ocular surface physiology with new LFU proteins may reveal interesting new biological mechanisms and help get to the heart of ocular surface dysfunction. PMID:18840430

EphA2 and Src regulate equatorial cell morphogenesis during lens development

PubMed Central

Cheng, Catherine; Ansari, Moham M.; Cooper, Jonathan A.; Gong, Xiaohua

2013-01-01

High refractive index and transparency of the eye lens require uniformly shaped and precisely aligned lens fiber cells. During lens development, equatorial epithelial cells undergo cell-to-cell alignment to form meridional rows of hexagonal cells. The mechanism that controls this morphogenesis from randomly packed cuboidal epithelial cells to highly organized hexagonal fiber cells remains unknown. In Epha2-/- mouse lenses, equatorial epithelial cells fail to form precisely aligned meridional rows; moreover, the lens fulcrum, where the apical tips of elongating epithelial cells constrict to form an anchor point before fiber cell differentiation and elongation at the equator, is disrupted. Phosphorylated Src-Y424 and cortactin-Y466, actin and EphA2 cluster at the vertices of wild-type hexagonal epithelial cells in organized meridional rows. However, phosphorylated Src and phosphorylated cortactin are not detected in disorganized Epha2-/- cells with altered F-actin distribution. E-cadherin junctions, which are normally located at the basal-lateral ends of equatorial epithelial cells and are diminished in newly differentiating fiber cells, become widely distributed in the apical, lateral and basal sides of epithelial cells and persist in differentiating fiber cells in Epha2-/- lenses. Src-/- equatorial epithelial cells also fail to form precisely aligned meridional rows and lens fulcrum. These results indicate that EphA2/Src signaling is essential for the formation of the lens fulcrum. EphA2 also regulates Src/cortactin/F-actin complexes at the vertices of hexagonal equatorial cells for cell-to-cell alignment. This mechanistic information explains how EphA2 mutations lead to disorganized lens cells that subsequently contribute to altered refractive index and cataracts in humans and mice. PMID:24026120

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

PubMed

Doğan, Ayşegül; Demirci, Selami; Şahin, Fikrettin

2015-01-01

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

Cytotoxic effects of octenidine mouth rinse on human fibroblasts and epithelial cells - an in vitro study.

PubMed

Schmidt, J; Zyba, V; Jung, K; Rinke, S; Haak, R; Mausberg, R F; Ziebolz, D

2016-01-01

This study compared the cytotoxicity of a new octenidine mouth rinse (MR) against gingival fibroblasts and epithelial cells with different established MRs. The following MRs were used: Octenidol (OCT), Chlorhexidine 0.2% (CHX), Listerine (LIS), Meridol (MER), Betaisodona (BET); and control (medium only). Human primary gingiva fibroblasts and human primary nasal epithelial cells were cultivated in cell-specific media (2 × 10(5) cells/ml) and treated with MR for 1, 5, and 15 min. Each test was performed 12 times. Metabolism activity was measured using a cytotoxicity assay. A cellometer analyzed cell viability, cell number, and cell diameter. The data were analyzed by two-way analysis of variance with subsequent Dunnett's test and additional t-tests. The cytotoxic effects of all MRs on fibroblasts and epithelial cells compared to the control depended on the contact time (p < 0.001). OCT and BET showed less influence on cell metabolism in fibroblasts than other MRs. OCT also demonstrated comparable but not significant results in epithelial cells (p > 0.005). Cell numbers of both cell types at all contact times revealed that OCT showed a less negative effect (p > 0.005), especially for epithelial cells compared to CHX after 15 min (p < 0.005). OCT and BET showed the best results for viability in fibroblasts (p > 0.005), but MER showed less influence than OCT in epithelial cells (p < 0.005). OCT is a potential alternative to CHX regarding cytotoxicity because of its lower cell-toxic effect against fibroblasts and epithelial cells.

Fluorescent in situ hybridization (FISH) on corneal impression cytology specimens (CICS): study of epithelial cell survival after keratoplasty.

PubMed

Catanese, Muriel; Popovici, Cornel; Proust, Hélène; Hoffart, Louis; Matonti, Frédéric; Cochereau, Isabelle; Conrath, John; Gabison, Eric E

2011-02-22

To assess corneal epithelial cell survival after keratoplasty. Corneal impression cytology (CIC) was performed on sex-mismatched corneal transplants. Fluorescent in situ hybridization (FISH) with sex chromosome-specific probes was performed to identify epithelial cell mosaicism and therefore allocate the donor or recipient origin of the cells. Twenty-four samples of corneal epithelial cells derived from 21 transplanted patients were analyzed. All patients received post-operative treatment using dexamethasone eye drops, with progressive tapering over 18 months, and nine patients also received 2% cyclosporine eye drops. Out of the 24 samples reaching quality criteria, sex mosaicism was found in 13, demonstrating the presence of donor-derived cells at the center of the graft for at least 211 days post keratoplasty. Kaplan-Meier analysis established a median survival of donor corneal epithelial cells of 385 days. Although not statistically significant, the disappearance of donor cells seemed to be delayed and the average number of persistent cells appeared to be greater when 2% cyclosporine was used topically as an additional immunosuppressive therapy. The combination of corneal impressions and FISH analysis is a valuable tool with negligible side effects to investigate the presence of epithelial cell mosaicism in sex-mismatched donor transplants. Epithelial cells survived at the center of the graft with a median survival of more than one year, suggesting slower epithelial turnover than previously described.

GM-CSF produced by non-hematopoietic cells is required for early epithelial cell proliferation and repair of injured colonic mucosa1,2

PubMed Central

Egea, Laia; McAllister, Christopher S.; Lakhdari, Omar; Minev, Ivelina; Shenouda, Steve; Kagnoff, Martin F.

2012-01-01

GM-CSF is a growth factor that promotes the survival and activation of macrophages and granulocytes, and dendritic cell (DC) differentiation and survival in vitro. The mechanism by which exogenous GM-CSF ameliorates the severity of Crohn’s disease in humans and colitis in murine models has been considered mainly to reflect its activity on myeloid cells. We used GM-CSF deficient (GM-CSF−/−) mice to probe the functional role of endogenous host-produced GM-CSF in a colitis model induced after injury to the colon epithelium. Dextran sodium sulfate (DSS) at doses that resulted in little epithelial damage and mucosal ulceration in wild type (WT) mice resulted in marked colon ulceration and delayed ulcer healing in GM-CSF−/− mice. Colon crypt epithelial cell proliferation in vivo was significantly decreased in GM-CSF−/− mice at early times after DSS injury. This was paralleled by decreased expression of crypt epithelial cell genes involved in cell cycle, proliferation, and wound healing. Decreased crypt cell proliferation and delayed ulcer healing in GM-CSF−/− mice were rescued by exogenous GM-CSF, indicating the lack of a developmental abnormality in the epithelial cell proliferative response in those mice. Non-hematopoietic cells and not myeloid cells produced the GM-CSF important for colon epithelial proliferation after DSS-induced injury as revealed by bone marrow chimera and DC depletion experiments, with colon epithelial cells being the cellular source of GM-CSF. Endogenous epithelial cell produced GM-CSF has a novel non-redundant role in facilitating epithelial cell proliferation and ulcer healing in response to injury of the colon crypt epithelium. PMID:23325885

Inhibition of 5-alpha-reductase activity induces stromal remodeling and smooth muscle de-differentiation in adult gerbil ventral prostate.

PubMed

Corradi, Lara S; Góes, Rejane M; Carvalho, Hernandes F; Taboga, Sebastião R

2004-06-01

Prostatic differentiation during embryogenesis and its further homeostatic state maintenance during adult life depend on androgens. Dihydrotestosterone, which is synthesized from testosterone by 5 alpha-reductase (5 alpha-r), is the active molecule triggering androgen action within the prostate. In the present work, we examined the effects of 5 alpha-reductase inhibition by finasteride in the ventral prostate (VP) of the adult gerbil, employing histochemical and electron microscopy techniques to demonstrate the morphological and organizational changes of the organ. After 10 days of finasteride treatment at a dose of 100 mg/kg/day, the prostatic complex (VP and dorsolateral prostate) absolute weight was reduced to about 18%. The epithelial cells became short and cuboidal, with less secretory blebs and reduced acid phosphatase activity. The luminal sectional area diminished, suggestive of decreased secretory activity. The stromal/epithelial ratio increased, the stroma becoming thicker but less cellular. There was a striking accumulation of collagen fibrils, which was accompanied by an increase in deposits of amorphous granular material adjacent to the basal lamina and in the clefts between smooth muscle cells (SMC). Additionally, the periacinar smooth muscle became loosely packed. Some SMC were atrophic and showed a denser array of the cytoskeleton, whereas other SMC had a highly irregular outline with numerous spine-like projections. The present data indicate that 5 alpha-r inhibition causes epithelial and stromal changes by affecting intra-prostatic hormone levels. These alterations are probably the result of an imbalance of the homeostatic interaction between the epithelium and the underlying stroma.

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

PubMed Central

Lukic, Ana; Ji, Jie; Idborg, Helena; Samuelsson, Bengt; Palmberg, Lena

2016-01-01

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

Coreceptors and Their Ligands in Epithelial γδ T Cell Biology

PubMed Central

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

2018-01-01

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

Cytokine Expression and Production by Purified Helicobacter pylori Urease in Human Gastric Epithelial Cells

PubMed Central

Tanahashi, Toshihito; Kita, Masakazu; Kodama, Tadashi; Yamaoka, Yoshio; Sawai, Naoki; Ohno, Tomoyuki; Mitsufuji, Shoji; Wei, Ya-Ping; Kashima, Kei; Imanishi, Jiro

2000-01-01

Cytokines have been proposed to play an important role in Helicobacter pylori-associated gastroduodenal diseases, but the exact mechanism of the cytokine induction remains unclear. H. pylori urease, a major component of the soluble proteins extracted from bacterial cells, is considered to be one of the virulence factors for the inflammation in the gastric mucosa that is produced in H. pylori infection. However, the response of human gastric epithelial cells to the stimulation of urease has not been investigated. In the present study, we used human gastric epithelial cells in a primary culture system and examined whether H. pylori urease stimulates the gastric epithelial cells to induce proinflammatory cytokines by reverse transcription-PCR and enzyme-linked immunosorbent assay. First, by using peripheral blood mononuclear cells (PBMC) and a gastric cancer cell line (MKN-45 cells), we confirmed the ability of purified H. pylori urease to induce the production of proinflammatory cytokines. Furthermore, we demonstrated that the human gastric epithelial cells produced interleukin-6 (IL-6) and tumor necrosis factor alpha, but not IL-8, following stimulation with purified urease. The patterns of cytokine induction differed among human PBMC, MKN-45 cells, and human gastric epithelial cells. These results suggest that the human gastric epithelial cells contribute to the induction of proinflammatory cytokines by the stimulation of H. pylori urease, indicating that the epithelial cells were involved in the mucosal inflammation that accompanied H. pylori infection. PMID:10639431

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

PubMed Central

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

2016-01-01

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

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

Rheinanthrone, a metabolite of sennoside A, triggers macrophage activation to decrease aquaporin-3 expression in the colon, causing the laxative effect of rhubarb extract.

PubMed

Kon, Risako; Ikarashi, Nobutomo; Nagoya, Chika; Takayama, Tomoko; Kusunoki, Yoshiki; Ishii, Makoto; Ueda, Harumi; Ochiai, Wataru; Machida, Yoshiaki; Sugita, Kazuyuki; Sugiyama, Kiyoshi

2014-02-27

Aquaporin-3 (AQP3) is expressed in mucosal epithelial cells in the colon and is important for regulating fecal water content. We examined the role of AQP3 in the laxative effect of rhubarb extract. After orally administering rhubarb extract or its major component (sennoside A) to rats, the fecal water content, AQP3 expression and prostaglandin E2 (PGE2) concentrations in the colon were examined. The mechanism by which sennoside A decreases the expression of AQP3 was examined using the human colon cancer HT-29 cells and macrophage-derived Raw264.7 cells. During diarrhea by rhubarb extract administration, the PGE2 levels in the colon increased while the AQP3 expression significantly decreased. Similar changes were also observed when sennoside A was administered. When sennoside A or its metabolites, rheinanthrone and rhein were added to Raw264.7 cells, a significant increase in the PGE2 concentration was observed only in cells treated with rheinanthrone. Fifteen minutes after adding PGE2 to the HT-29 cells, the AQP3 expression decreased to approximately 40% of the control. When pretreated with indomethacin, sennoside A neither decreased the AQP3 expression nor induced diarrhea. Sennoside A may decrease AQP3 expression in the colon to inhibit water transport from the luminal to the vascular side, leading to a laxative effect. The decreases in the levels of AQP3 are caused by rheinanthrone, which is a metabolite of sennoside A, this metabolite activates the macrophages in the colon and increases the secretion of PGE2; PGE2 acts as a paracrine factor and decreases AQP3 expression in colon mucosal epithelial cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Role of Intestinal Epithelial Cells in Immune Effects Mediated by Gram-Positive Probiotic Bacteria: Involvement of Toll-Like Receptors

PubMed Central

Vinderola, Gabriel; Matar, Chantal; Perdigon, Gabriela

2005-01-01

The mechanisms by which probiotic bacteria exert their effects on the immune system are not completely understood, but the epithelium may be a crucial player in the orchestration of the effects induced. In a previous work, we observed that some orally administered strains of lactic acid bacteria (LAB) increased the number of immunoglobulin A (IgA)-producing cells in the small intestine without a concomitant increase in the CD4+ T-cell population, indicating that some LAB strains induce clonal expansion only of B cells triggered to produce IgA. The present work aimed to study the cytokines induced by the interaction of probiotic LAB with murine intestinal epithelial cells (IEC) in healthy animals. We focused our investigation mainly on the secretion of interleukin 6 (IL-6) necessary for the clonal expansion of B cells previously observed with probiotic bacteria. The role of Toll-like receptors (TLRs) in such interaction was also addressed. The cytokines released by primary cultures of IEC in animals fed with Lactobacillus casei CRL 431 or Lactobacillus helveticus R389 were determined. Cytokines were also determined in the supernatants of primary cultures of IEC of unfed animals challenged with different concentrations of viable or nonviable lactobacilli and Escherichia coli, previously blocked or not with anti-TLR2 and anti-TLR4. We concluded that the small intestine is the place where a major distinction would occur between probiotic LAB and pathogens. This distinction comprises the type of cytokines released and the magnitude of the response, cutting across the line that separates IL-6 necessary for B-cell differentiation, which was the case with probiotic lactobacilli, from inflammatory levels of IL-6 for pathogens. PMID:16148174

In vitro response of retinal pigment epithelial cells exposed to chitosan materials prepared with different cross-linkers.

PubMed

Lai, Jui-Yang; Li, Ya-Ting; Wang, Tsu-Pin

2010-01-01

The interaction between cells and biopolymers is the evaluation indicator of the biocompatibility of materials. The purpose of this work was to examine the responses of retinal pigment epithelial (RPE) cells to genipin (GP) or glutaraldehyde (GTA) cross-linked chitosan by means of cell viability assays, cytokine expression analyses, and apoptosis assays. Evaluations of non-cross-linked chitosan were conducted simultaneously for comparison. Both GP and GTA treated samples with the same extent of cross-linking (around 80%) were prepared by varying cross-linking time. Our results showed that GP cross-linking was carried out by either radical polymerization of the monomers or S(N)2 nucleophilic substitution reaction involving the replacement of the ester group on the monomer with a secondary amide linkage. On the other hand, GTA could react with free amino groups of chitosan, leading to the formation of either the Schiff bases or the Michael-type adducts with terminal aldehydes. The biocompatibility of non-cross-linked chitosan membranes was demonstrated by the absence of any signs of toxicity or inflammation reaction. The present study showed that the ARPE-19 cells exposed to GTA cross-linked chitosan membranes had significantly higher cytotoxicity, interleukin-6 levels, and number of TUNEL-positive nuclei than did those exposed to GP treated samples. In addition, the materials modified with GTA trigger apoptosis at an early stage and may induce toxicity in the RPE cells later. The findings suggest that while the chitosan molecules bridged by GP are satisfactorily cytocompatible, the counterparts treated by GTA do not seem to be tolerated. In terms of material safety, the GP cross-linked chitosan may be compatible with human RPE cells and may have a potential application as delivery carriers in the treatment of posterior segment diseases.

The Contribution of the Airway Epithelial Cell to Host Defense.

PubMed

Stanke, Frauke

2015-01-01

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

Role of medullary progenitor cells in epithelial cell migration and proliferation

PubMed Central

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

2014-01-01

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

Helicobacter pylori colonization ameliorates glucose homeostasis in mice through a PPAR γ-dependent mechanism.

PubMed

Bassaganya-Riera, Josep; Dominguez-Bello, Maria Gloria; Kronsteiner, Barbara; Carbo, Adria; Lu, Pinyi; Viladomiu, Monica; Pedragosa, Mireia; Zhang, Xiaoying; Sobral, Bruno W; Mane, Shrinivasrao P; Mohapatra, Saroj K; Horne, William T; Guri, Amir J; Groeschl, Michael; Lopez-Velasco, Gabriela; Hontecillas, Raquel

2012-01-01

There is an inverse secular trend between the incidence of obesity and gastric colonization with Helicobacter pylori, a bacterium that can affect the secretion of gastric hormones that relate to energy homeostasis. H. pylori strains that carry the cag pathogenicity island (PAI) interact more intimately with gastric epithelial cells and trigger more extensive host responses than cag(-) strains. We hypothesized that gastric colonization with H. pylori strains differing in cag PAI status exert distinct effects on metabolic and inflammatory phenotypes. To test this hypothesis, we examined metabolic and inflammatory markers in db/db mice and mice with diet-induced obesity experimentally infected with isogenic forms of H. pylori strain 26695: the cag PAI wild-type and its cag PAI mutant strain 99-305. H. pylori colonization decreased fasting blood glucose levels, increased levels of leptin, improved glucose tolerance, and suppressed weight gain. A response found in both wild-type and mutant H. pylori strain-infected mice included decreased white adipose tissue macrophages (ATM) and increased adipose tissue regulatory T cells (Treg) cells. Gene expression analyses demonstrated upregulation of gastric PPAR γ-responsive genes (i.e., CD36 and FABP4) in H. pylori-infected mice. The loss of PPAR γ in immune and epithelial cells in mice impaired the ability of H. pylori to favorably modulate glucose homeostasis and ATM infiltration during high fat feeding. Gastric infection with some commensal strains of H. pylori ameliorates glucose homeostasis in mice through a PPAR γ-dependent mechanism and modulates macrophage and Treg cell infiltration into the abdominal white adipose tissue.

Pseudomonas aeruginosa proteolytically alters the interleukin 22-dependent lung mucosal defense.

PubMed

Guillon, Antoine; Brea, Deborah; Morello, Eric; Tang, Aihua; Jouan, Youenn; Ramphal, Reuben; Korkmaz, Brice; Perez-Cruz, Magdiel; Trottein, Francois; O'Callaghan, Richard J; Gosset, Philippe; Si-Tahar, Mustapha

2017-08-18

The IL-22 signaling pathway is critical for regulating mucosal defense and limiting bacterial dissemination. IL-22 is unusual among interleukins because it does not directly regulate the function of conventional immune cells, but instead targets cells at outer body barriers, such as respiratory epithelial cells. Consequently, IL-22 signaling participates in the maintenance of the lung mucosal barrier by controlling cell proliferation and tissue repair, and enhancing the production of specific chemokines and anti-microbial peptides. Pseudomonas aeruginosa is a major pathogen of ventilator-associated pneumonia and causes considerable lung tissue damage. A feature underlying the pathogenicity of this bacterium is its capacity to persist and develop in the host, particularly in the clinical context of nosocomial lung infections. We aimed to investigate the ability of P. auruginosa to disrupt immune-epithelial cells cross-talk. We found that P. aeruginosa escapes the host mucosal defenses by degrading IL-22, leading to severe inhibition of IL-22-mediated immune responses. We demonstrated in vitro that, protease IV, a type 2 secretion system-dependent serine protease, is responsible for the degradation of IL-22 by P. aeruginosa. Moreover, the major anti-proteases molecules present in the lungs were unable to inhibit protease IV enzymatic activity. In addition, tracheal aspirates of patients infected by P. aeruginosa contain protease IV activity which further results in IL-22 degradation. This so far undescribed cleavage of IL-22 by a bacterial protease is likely to be an immune-evasion strategy that contributes to P. aeruginosa-triggered respiratory infections.

Diverse mitotic functions of the cytoskeletal cross-linking protein Shortstop suggest a role in Dynein/Dynactin activity.

PubMed

Dewey, Evan B; Johnston, Christopher A

2017-09-15

Proper assembly and orientation of the bipolar mitotic spindle is critical to the fidelity of cell division. Mitotic precision fundamentally contributes to cell fate specification, tissue development and homeostasis, and chromosome distribution within daughter cells. Defects in these events are thought to contribute to several human diseases. The underlying mechanisms that function in spindle morphogenesis and positioning remain incompletely defined, however. Here we describe diverse roles for the actin-microtubule cross-linker Shortstop (Shot) in mitotic spindle function in Drosophila Shot localizes to mitotic spindle poles, and its knockdown results in an unfocused spindle pole morphology and a disruption of proper spindle orientation. Loss of Shot also leads to chromosome congression defects, cell cycle progression delay, and defective chromosome segregation during anaphase. These mitotic errors trigger apoptosis in Drosophila epithelial tissue, and blocking this apoptotic response results in a marked induction of the epithelial-mesenchymal transition marker MMP-1. The actin-binding domain of Shot directly interacts with Actin-related protein-1 (Arp-1), a key component of the Dynein/Dynactin complex. Knockdown of Arp-1 phenocopies Shot loss universally, whereas chemical disruption of F-actin does so selectively. Our work highlights novel roles for Shot in mitosis and suggests a mechanism involving Dynein/Dynactin activation. © 2017 Dewey and Johnston. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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

PubMed

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

2009-04-01

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

Niche-induced cell death and epithelial phagocytosis regulate hair follicle stem cell pool.

PubMed

Mesa, Kailin R; Rompolas, Panteleimon; Zito, Giovanni; Myung, Peggy; Sun, Thomas Y; Brown, Samara; Gonzalez, David G; Blagoev, Krastan B; Haberman, Ann M; Greco, Valentina

2015-06-04

Tissue homeostasis is achieved through a balance of cell production (growth) and elimination (regression). In contrast to tissue growth, the cells and molecular signals required for tissue regression remain unknown. To investigate physiological tissue regression, we use the mouse hair follicle, which cycles stereotypically between phases of growth and regression while maintaining a pool of stem cells to perpetuate tissue regeneration. Here we show by intravital microscopy in live mice that the regression phase eliminates the majority of the epithelial cells by two distinct mechanisms: terminal differentiation of suprabasal cells and a spatial gradient of apoptosis of basal cells. Furthermore, we demonstrate that basal epithelial cells collectively act as phagocytes to clear dying epithelial neighbours. Through cellular and genetic ablation we show that epithelial cell death is extrinsically induced through transforming growth factor (TGF)-β activation and mesenchymal crosstalk. Strikingly, our data show that regression acts to reduce the stem cell pool, as inhibition of regression results in excess basal epithelial cells with regenerative abilities. This study identifies the cellular behaviours and molecular mechanisms of regression that counterbalance growth to maintain tissue homeostasis.

Lateral adhesion drives reintegration of misplaced cells into epithelial monolayers.

PubMed

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

2015-11-01

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

Generation of Epithelial Cell Populations from Human Pluripotent Stem Cells Using a Small-Molecule Inhibitor of Src Family Kinases.

PubMed

Selekman, Joshua A; Lian, Xiaojun; Palecek, Sean P

2016-01-01

Human pluripotent stem cells (hPSCs), under the right conditions, can be engineered to generate populations of any somatic cell type. Knowledge of what mechanisms govern differentiation towards a particular lineage is often quite useful for efficiently producing somatic cell populations from hPSCs. Here, we have outlined a strategy for deriving populations of simple epithelial cells, as well as more mature epidermal keratinocyte progenitors, from hPSCs by exploiting a mechanism previously shown to direct epithelial differentiation of hPSCs. Specifically, we describe how to direct epithelial differentiation of hPSCs using an Src family kinase inhibitor, SU6656, which has been shown to modulate β-catenin translocation to the cell membrane and thus promote epithelial differentiation. The differentiation platform outlined here produces cells with the ability to terminally differentiate to epidermal keratinocytes in culture through a stable simple epithelial cell intermediate that can be expanded in culture for numerous (>10) passages.

Role of the epithelial cell rests of Malassez in the development, maintenance and regeneration of periodontal ligament tissues.

PubMed

Xiong, Jimin; Gronthos, Stan; Bartold, P Mark

2013-10-01

Periodontitis is a highly prevalent inflammatory disease that results in damage to the tooth-supporting tissues, potentially leading to tooth loss. Periodontal tissue regeneration is a complex process that involves the collaboration of two hard tissues (cementum and alveolar bone) and two soft tissues (gingiva and periodontal ligament). To date, no periodontal-regenerative procedures provide predictable clinical outcomes. To understand the rational basis of regenerative procedures, a better understanding of the events associated with the formation of periodontal components will help to establish reliable strategies for clinical practice. An important aspect of this is the role of the Hertwig's epithelial root sheath in periodontal development and that of its descendants, the epithelial cell rests of Malassez, in the maintenance of the periodontium. An important structure during tooth root development, the Hertwig's epithelial root sheath is not only a barrier between the dental follicle and dental papilla cells but is also involved in determining the shape, size and number of roots and in the development of dentin and cementum, and may act as a source of mesenchymal progenitor cells for cementoblasts. In adulthood, the epithelial cell rests of Malassez are the only odontogenic epithelial population in the periodontal ligament. Although there is no general agreement on the functions of the epithelial cell rests of Malassez, accumulating evidence suggests that the putative roles of the epithelial cell rests of Malassez in adult periodontal ligament include maintaining periodontal ligament homeostasis to prevent ankylosis and maintain periodontal ligament space, to prevent root resorption, to serve as a target during periodontal ligament innervation and to contribute to cementum repair. Recently, ovine epithelial cell rests of Malassez cells have been shown to harbor clonogenic epithelial stem-cell populations that demonstrate similar properties to mesenchymal stromal/stem cells, both functionally and phenotypically. Therefore, the epithelial cell rests of Malassez, rather than being 'cell rests', as indicated by their name, are an important source of stem cells that might play a pivotal role in periodontal regeneration. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

PubMed Central

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

2017-01-01

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

[Pathological and immunohistochemical analysis of giant cells of pancreas].

PubMed

Miyake, T; Suda, K; Yamamura, A; Tada, Y

1997-10-01

Multinucleated giant cells in the pancreas (five giant cell carcinomas, a mucinous cystadenocarcinoma attended with many osteoclast-like giant cells, 42 invasive ductal carcinomas and 29 chronic pancreatitises) were examined. Three types of multinucleated giant cell were identified: epithelial type, coexpressive type, mesenchymal type. Epithelial type expressed epithelial markers, such as keratin and EMA in 23 ductal carcinomas. Coexpressive type expressed both epithelial markers and mesenchymal marker vimentin was in four ductal carcinomas. Mesenchymal type expressed mesenchymal markers, vimentin and CD68 in four osteoclastoid type giant cell carcinomas, the mucinous cystadenocarcinoma, six ductal carcinomas and ten chronic pancreatitises. Epithelial and coexpressive type were considered to be epithelial neoplastic origin, those had bizarre appearance and transitional area from definite adenocarcinoma area. Vimentin expression is associated with sarcomatous proliferation. Mesenchymal type was considered to be nonneoplastic and a certain type of macrophage polykaryons.

Metformin and Chemotherapy in Treating Patients With Stage III-IV Ovarian, Fallopian Tube, or Primary Peritoneal Cancer

ClinicalTrials.gov

2018-04-17

Brenner Tumor; Malignant Ascites; Malignant Pleural Effusion; Ovarian Clear Cell Cystadenocarcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Mixed Epithelial Carcinoma; Ovarian Serous Cystadenocarcinoma; Ovarian Undifferentiated Adenocarcinoma; Recurrent Fallopian Tube Cancer; Recurrent Ovarian Epithelial Cancer; Recurrent Ovarian Germ Cell Tumor; Recurrent Primary Peritoneal Cavity Cancer; Stage IIIA Fallopian Tube Cancer; Stage IIIA Ovarian Epithelial Cancer; Stage IIIA Ovarian Germ Cell Tumor; Stage IIIA Primary Peritoneal Cavity Cancer; Stage IIIB Fallopian Tube Cancer; Stage IIIB Ovarian Epithelial Cancer; Stage IIIB Ovarian Germ Cell Tumor; Stage IIIB Primary Peritoneal Cavity Cancer; Stage IIIC Fallopian Tube Cancer; Stage IIIC Ovarian Epithelial Cancer; Stage IIIC Ovarian Germ Cell Tumor; Stage IIIC Primary Peritoneal Cavity Cancer; Stage IV Fallopian Tube Cancer; Stage IV Ovarian Epithelial Cancer; Stage IV Ovarian Germ Cell Tumor; Stage IV Primary Peritoneal Cavity Cancer

Urokinase and the intestinal mucosa: evidence for a role in epithelial cell turnover

PubMed Central

Gibson, P; Birchall, I; Rosella, O; Albert, V; Finch, C; Barkla, D; Young, G

1998-01-01

Background—The functions of urokinase in intestinal epithelia are unknown. 
Aims—To determine the relation of urokinase expressed by intestinal epithelial cells to their position in the crypt-villus/surface axis and of mucosal urokinase activity to epithelial proliferative kinetics in the distal colon. 
Methods—Urokinase expression was examined immunohistochemically in human intestinal mucosa. Urokinase activity was measured colorimetrically in epithelial cells isolated sequentially from the crypt-villus axis of the rat small intestine. In separate experiments, urokinase activity and epithelial kinetics (measured stathmokinetically) were measured in homogenates of distal colonic mucosa of 14 groups of eight rats fed diets known to alter epithelial turnover. 
Results—From the crypt base, an ascending gradient of expression and activity of urokinase was associated with the epithelial cells. Median mucosal urokinase activities in each of the dietary groups of rats correlated positively with autologous median number of metaphase arrests per crypt (r=0.68; p<0.005) and per 100 crypt cells (r=0.75; p<0.001), but not with crypt column height. 
Conclusions—Localisation of an enzyme capable of leading to digestion of cell substratum in the region where cells are loosely attached to their basement membrane, and the association of its activity with indexes of cell turnover, suggest a role for urokinase in facilitating epithelial cell loss in the intestine. 

 Keywords: urokinase; intestinal epithelium; colon; epithelial proliferation PMID:9824347

AKT1 provides an essential survival signal required for differentiation and stratification of primary human keratinocytes.

PubMed

Thrash, Barry R; Menges, Craig W; Pierce, Robert H; McCance, Dennis J

2006-04-28

Keratinocyte differentiation and stratification are complex processes involving multiple signaling pathways, which convert a basal proliferative cell into an inviable rigid squame. Loss of attachment to the basement membrane triggers keratinocyte differentiation, while in other epithelial cells, detachment from the extracellular matrix leads to rapid programmed cell death or anoikis. The potential role of AKT in providing a survival signal necessary for stratification and differentiation of primary human keratinocytes was investigated. AKT activity increased during keratinocyte differentiation and was attributed to the specific activation of AKT1 and AKT2. Targeted reduction of AKT1 expression, but not AKT2, by RNA interference resulted in an abnormal epidermis in organotypic skin cultures with a thin parabasal region and a pronounced but disorganized cornified layer. This abnormal stratification was due to significant cell death in the suprabasal layers and was alleviated by caspase inhibition. Normal expression patterns of both early and late markers of keratinocyte differentiation were also disrupted, producing a poorly developed stratum corneum.

Use of lectin-functionalized particles for oral immunotherapy

PubMed Central

Diesner, Susanne C; Wang, Xue-Yan; Jensen-Jarolim, Erika; Untersmayr, Eva; Gabor, Franz

2013-01-01

Immunotherapy, in recent times, has found its application in a variety of immunologically mediated diseases. Oral immunotherapy may not only increase patient compliance but may, in particular, also induce both systemic as well as mucosal immune responses, due to mucosal application of active agents. To improve the bioavailability and to trigger strong immunological responses, recent research projects focused on the encapsulation of drugs and antigens into polymer particles. These particles protect the loaded antigen from the harsh conditions in the GI tract. Furthermore, modification of the surface of particles by the use of lectins, such as Aleuria aurantia lectin, wheatgerm agglutinin or Ulex europaeus-I, enhances the binding to epithelial cells, in particular to membranous cells, of the mucosa-associated lymphoid tissue. Membranous cell-specific targeting leads to an improved transepithelial transport of the particle carriers. Thus, enhanced uptake and presentation of the encapsulated antigen by antigen-presenting cells favor strong systemic, but also local, mucosal immune responses. PMID:22834202

NF-Y loss triggers p53 stabilization and apoptosis in HPV18-positive cells by affecting E6 transcription.

PubMed

Benatti, Paolo; Basile, Valentina; Dolfini, Diletta; Belluti, Silvia; Tomei, Margherita; Imbriano, Carol

2016-07-19

The expression of the high risk HPV18 E6 and E7 oncogenic proteins induces the transformation of epithelial cells, through the disruption of p53 and Rb function. The binding of cellular transcription factors to cis-regulatory elements in the viral Upstream Regulatory Region (URR) stimulates E6/E7 transcription. Here, we demonstrate that the CCAAT-transcription factor NF-Y binds to a non-canonical motif within the URR and activates viral gene expression. In addition, NF-Y indirectly up-regulates HPV18 transcription through the transactivation of multiple cellular transcription factors. NF-YA depletion inhibits the expression of E6 and E7 genes and re-establishes functional p53. The activation of p53 target genes in turn leads to apoptotic cell death. Finally, we show that NF-YA loss sensitizes HPV18-positive cells toward the DNA damaging agent Doxorubicin, via p53-mediated transcriptional response.

Epithelial junctions, cytoskeleton, and polarity.

PubMed

Pásti, Gabriella; Labouesse, Michel

2014-11-04

A distinctive feature of polarized epithelial cells is their specialized junctions, which contribute to cell integrity and provide platforms to orchestrate cell shape changes. This chapter discusses the composition, assembly and remodeling of C. elegans cell-cell (CeAJ) and hemidesmosome-like cell-extracellular matrix junctions (CeHD), proteins that anchor the cytoskeleton, and mechanisms involved in establishing epithelial polarity. Major recent progress in this area has come from the analysis of mechanisms that maintain cell polarity, which involve lipids and trafficking, and on the impact of mechanical forces on junction remodeling. This chapter focuses on cellular, rather than developmental, aspects of epithelial cells.

Activation of neurokinin-1 receptors during ozone inhalation contributes to epithelial injury and repair.

PubMed

Oslund, Karen L; Hyde, Dallas M; Putney, Leialoha F; Alfaro, Mario F; Walby, William F; Tyler, Nancy K; Schelegle, Edward S

2008-09-01

We investigated the importance of neurokinin (NK)-1 receptors in epithelial injury and repair and neutrophil function. Conscious Wistar rats were exposed to 1 ppm ozone or filtered air for 8 hours, followed by an 8-hour postexposure period. Before exposure, we administered either the NK-1 receptor antagonist, SR140333, or saline as a control. Ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, whole mounts of airway dissected lung lobes were immunostained for 5-bromo-2'-deoxyuridine, a marker of epithelial proliferation. Both ethidium homodimer and 5-bromo-2'-deoxyuridine-positive epithelial cells were quantified in specific airway generations. Rats treated with the NK-1 receptor antagonist had significantly reduced epithelial injury and epithelial proliferation compared with control rats. Sections of terminal bronchioles showed no significant difference in the number of neutrophils in airways between groups. In addition, staining ozone-exposed lung sections for active caspase 3 showed no apoptotic cells, but ethidium-positive cells colocalized with the orphan nuclear receptor, Nur77, a marker of nonapoptotic, programmed cell death mediated by the NK-1 receptor. An immortalized human airway epithelial cell line, human bronchial epithelial-1, showed no significant difference in the number of oxidant stress-positive cells during exposure to hydrogen peroxide and a range of SR140333 doses, demonstrating no antioxidant effect of the receptor antagonist. We conclude that activation of the NK-1 receptor during acute ozone inhalation contributes to epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways.

Immunobiotics for the Bovine Host: Their Interaction with Intestinal Epithelial Cells and Their Effect on Antiviral Immunity

PubMed Central

Villena, Julio; Aso, Hisashi; Rutten, Victor P. M. G.; Takahashi, Hideki; van Eden, Willem; Kitazawa, Haruki

2018-01-01

The scientific community has reported several cases of microbes that exhibit elevated rates of antibiotic resistance in different regions of the planet. Due to this emergence of antimicrobial resistant microorganisms, the use of antibiotics as promoters of livestock animals’ growth is being banned in most countries around the world. One of the challenges of agricultural immunology therefore is to find alternatives by modulating the immune system of animals in drug-independent safe food production systems. In this regard, in an effort to supplant antibiotics from bovine feeds, several alternatives were proposed including the use of immunomodulatory probiotics (immunobiotics). The purpose of this review is to provide an update of the status of the modulation of intestinal antiviral innate immunity of the bovine host by immunobiotics, and the beneficial impact of immunobiotics on viral infections, focused on intestinal epithelial cells (IECs). The results of our group, which demonstrate the capacity of immunobiotic strains to beneficially modulate Toll-like receptor 3-triggered immune responses in bovine IECs and improve the resistance to viral infections, are highlighted. This review provides comprehensive information on the innate immune response of bovine IECs against virus, which can be further investigated for the development of strategies aimed to improve defenses in the bovine host. PMID:29599767

Modelling staphylococcal pneumonia in a human 3D lung tissue model system delineates toxin-mediated pathology

PubMed Central

Mairpady Shambat, Srikanth; Chen, Puran; Nguyen Hoang, Anh Thu; Bergsten, Helena; Vandenesch, Francois; Siemens, Nikolai; Lina, Gerard; Monk, Ian R.; Foster, Timothy J.; Arakere, Gayathri; Svensson, Mattias; Norrby-Teglund, Anna

2015-01-01

ABSTRACT Staphylococcus aureus necrotizing pneumonia is recognized as a toxin-mediated disease, yet the tissue-destructive events remain elusive, partly as a result of lack of mechanistic studies in human lung tissue. In this study, a three-dimensional (3D) tissue model composed of human lung epithelial cells and fibroblasts was used to delineate the role of specific staphylococcal exotoxins in tissue pathology associated with severe pneumonia. To this end, the models were exposed to the mixture of exotoxins produced by S. aureus strains isolated from patients with varying severity of lung infection, namely necrotizing pneumonia or lung empyema, or to purified toxins. The necrotizing pneumonia strains secreted high levels of α-toxin and Panton-Valentine leukocidin (PVL), and triggered high cytotoxicity, inflammation, necrosis and loss of E-cadherin from the lung epithelium. In contrast, the lung empyema strain produced moderate levels of PVL, but negligible amounts of α-toxin, and triggered limited tissue damage. α-toxin had a direct damaging effect on the epithelium, as verified using toxin-deficient mutants and pure α-toxin. Moreover, PVL contributed to pathology through the lysis of neutrophils. A combination of α-toxin and PVL resulted in the most severe epithelial injury. In addition, toxin-induced release of pro-inflammatory mediators from lung tissue models resulted in enhanced neutrophil migration. Using a collection of 31 strains from patients with staphylococcal pneumonia revealed that strains producing high levels of α-toxin and PVL were cytotoxic and associated with fatal outcome. Also, the strains that produced the highest toxin levels induced significantly greater epithelial disruption. Of importance, toxin-mediated lung epithelium destruction could be inhibited by polyspecific intravenous immunoglobulin containing antibodies against α-toxin and PVL. This study introduces a novel model system for study of staphylococcal pneumonia in a human setting. The results reveal that the combination and levels of α-toxin and PVL correlate with tissue pathology and clinical outcome associated with pneumonia. PMID:26398950

Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells

PubMed Central

Bhattacharya, Sujoy; Ray, Ramesh M.; Johnson, Leonard R.

2014-01-01

Homeostasis of the gastrointestinal epithelium is dependent upon a balance between cell proliferation and apoptosis. Cyclin-dependent kinases (Cdks) are well known for their role in cell proliferation. Previous studies from our group have shown that polyamine-depletion of intestinal epithelial cells (IEC-6) decreases cyclin-dependent kinase 2 (Cdk2) activity, increases p53 and p21Cip1 protein levels, induces G1 arrest, and protects cells from camptothecin (CPT)-induced apoptosis. Although emerging evidence suggests that members of the Cdk family are involved in the regulation of apoptosis, their roles directing apoptosis of IEC-6 cells are not known. In this study, we report that inhibition of Cdk1, 2, and 9 (with the broad range Cdk inhibitor, AZD5438) in proliferating IEC-6 cells triggered DNA damage, activated p53 signaling, inhibited proliferation, and induced apoptosis. By contrast, inhibition of Cdk2 (with NU6140) increased p53 protein and activity, inhibited proliferation, but had no effect on apoptosis. Notably, AZD5438 sensitized, whereas, NU6140 rescued proliferating IEC-6 cells from CPT-induced apoptosis. However, in colon carcinoma (Caco2) cells with mutant p53, treatment with either AZD5438 or NU6140 blocked proliferation, albeit more robustly with AZD5438. Both Cdk inhibitors induced apoptosis in Caco2 cells in a p53-independent manner. In serum starved quiescent IEC-6 cells, both AZD5438 and NU6140 decreased TNF- /CPT-induced activation of p53 and, consequently, rescued cells from apoptosis, indicating that sustained Cdk activity is required for apoptosis of quiescent cells. Furthermore, AZD5438 partially reversed the protective effect of polyamine depletion whereas NU6140 had no effect. Together, these results demonstrate that Cdks possess opposing roles in the control of apoptosis in quiescent and proliferating cells. In addition, Cdk inhibitors uncouple proliferation from apoptosis in a p53-dependent manner. PMID:24242917

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

PubMed Central

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

2016-01-01

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

Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells.

PubMed

Danelishvili, Lia; McGarvey, Jeffery; Li, Yong-Jun; Bermudez, Luiz E

2003-09-01

Mycobacterium tuberculosis interacts with macrophages and epithelial cells in the alveolar space of the lung, where it is able to invade and replicate in both cell types. M. tuberculosis-associated cytotoxicity to these cells has been well documented, but the mechanisms of host cell death are not well understood. We examined the induction of apoptosis and necrosis of human macrophages (U937) and type II alveolar epithelial cells (A549) by virulent (H37Rv) and attenuated (H37Ra) M. tuberculosis strains. Apoptosis was determined by both enzyme-linked immunosorbent assay (ELISA) and TdT-mediated dUTP nick end labelling (TUNEL) assay, whereas necrosis was evaluated by the release of lactate dehydrogenase (LDH). Both virulent and attenuated M. tuberculosis induced apoptosis in macrophages; however, the attenuated strain resulted in significantly more apoptosis than the virulent strain after 5 days of infection. In contrast, cytotoxicity of alveolar cells was the result of necrosis, but not apoptosis. Although infection with M. tuberculosis strains resulted in apoptosis of 14% of the cells on the monolayer, cell death associated with necrosis was observed in 59% of alveolar epithelial cells after 5 days of infection. Infection with M. tuberculosis suppressed apoptosis of alveolar epithelial cells induced by the kinase inhibitor, staurosporine. Because our findings suggest that M. tuberculosis can modulate the apoptotic response of macrophages and epithelial cells, we carried out an apoptosis pathway-specific cDNA microarray analysis of human macrophages and alveolar epithelial cells. Whereas the inhibitors of apoptosis, bcl-2 and Rb, were upregulated over 2.5-fold in infected (48 h) alveolar epithelial cells, the proapoptotic genes, bad and bax, were downregulated. The opposite was observed when U937 macrophages were infected with M. tuberculosis. Upon infection of alveolar epithelial cells with M. tuberculosis, the generation of apoptosis, as determined by the expression of caspase-1, caspase-3 and caspase-10, was inhibited. Inhibition of replication of intracellular bacteria resulted in an increase in apoptosis in both cell types. Our results showed that the differential induction of apoptosis between macrophages and alveolar epithelial cells represents specific strategies of M. tuberculosis for survival in the host.

NK cells are necessary for recovery of corneal CD11c+ dendritic cells after epithelial abrasion injury

USDA-ARS?s Scientific Manuscript database

Mechanisms controlling CD11c(+) MHCII(+) DCs during corneal epithelial wound healing were investigated in a murine model of corneal abrasion. Selective depletion of NKp46(+) CD3- NK cells that normally migrate into the cornea after epithelial abrasion resulted in >85% reduction of the epithelial CD1...

Establishment and characterization of a lactating dairy goat mammary gland epithelial cell line.

PubMed

Tong, Hui-Li; Li, Qing-Zhang; Gao, Xue-Jun; Yin, De-Yun

2012-03-01

To study milk synthesis in dairy goat mammary gland, we had established an in vitro lactating dairy goat mammary epithelial cell (DGMEC) line. Mammary tissues of Guan Zhong dairy goats at 35 d of lactation were dispersed and cultured in a medium containing epithelial growth factor, insulin-like growth factor-1, insulin transferrin serum, and fetal bovine serum. Epithelial cells were enriched by digesting with 0.25% trypsin repeatedly to remove fibroblast cells and were identified as epithelial origin by staining with antibody against cytokeratine 18. The DGMECs displayed monolayer, cobble-stone, epithelial-like morphology, and formed alveoli-like structures and island monolayer aggregates which were the typical characteristics of mammary epithelial cells. A one-half logarithmically growth curve and cytoplasmic lipid droplets in these cells were observed. In this paper, we also studied the lactating function of DGMECs. Results showed that DGMECs could secrete lactose and β-casein. Lactating function of the cells had no obvious change after 48 h treated by insulin, while prolactin could obviously raise the secretion of milk proteins and lactose.

Infection of human intestinal epithelial cells with invasive bacteria upregulates apical intercellular adhesion molecule-1 (ICAM)-1) expression and neutrophil adhesion.

PubMed Central

Huang, G T; Eckmann, L; Savidge, T C; Kagnoff, M F

1996-01-01

The acute host response to gastrointestinal infection with invasive bacteria is characterized by an accumulation of neutrophils in the lamina propria, and neutrophil transmigration to the luminal side of the crypts. Intestinal epithelial cells play an important role in the recruitment of inflammatory cells to the site of infection through the secretion of chemokines. However, little is known regarding the expression, by epithelial cells, of molecules that are involved in interactions between the epithelium and neutrophils following bacterial invasion. We report herein that expression of ICAM-1 on human colon epithelial cell lines, and on human enterocytes in an in vivo model system, is upregulated following infection with invasive bacteria. Increased ICAM-1 expression in the early period (4-9 h) after infection appeared to result mainly from a direct interaction between invaded bacteria and host epithelial cells since it co-localized to cells invaded by bacteria, and the release of soluble factors by epithelial cells played only a minor role in mediating increased ICAM-1 expression. Furthermore, ICAM-1 was expressed on the apical side of polarized intestinal epithelial cells, and increased expression was accompanied by increased neutrophil adhesion to these cells. ICAM-1 expression by intestinal epithelial cells following infection with invasive bacteria may function to maintain neutrophils that have transmigrated through the epithelium in close contact with the intestinal epithelium, thereby reducing further invasion of the mucosa by invading pathogens. PMID:8755670

Hypothiocyanite produced by human and rat respiratory epithelial cells inactivates extracellular H1N2 influenza A virus.

PubMed

Gingerich, Aaron; Pang, Lan; Hanson, Jarod; Dlugolenski, Daniel; Streich, Rebecca; Lafontaine, Eric R; Nagy, Tamás; Tripp, Ralph A; Rada, Balázs

2016-01-01

Our aim was to study whether an extracellular, oxidative antimicrobial mechanism inherent to tracheal epithelial cells is capable of inactivating influenza H1N2 virus. Epithelial cells were isolated from tracheas of male Sprague-Dawley rats. Both primary human and rat tracheobronchial epithelial cells were differentiated in air-liquid interface cultures. A/swine/Illinois/02860/09 (swH1N2) influenza A virions were added to the apical side of airway cells for 1 h in the presence or absence of lactoperoxidase or thiocyanate. Characterization of rat epithelial cells (morphology, Duox expression) occurred via western blotting, PCR, hydrogen peroxide production measurement and histology. The number of viable virions was determined by plaque assays. Statistical difference of the results was analyzed by ANOVA and Tukey's test. Our data show that rat tracheobronchial epithelial cells develop a differentiated, polarized monolayer with high transepithelial electrical resistance, mucin production and expression of dual oxidases. Influenza A virions are inactivated by human and rat epithelial cells via a dual oxidase-, lactoperoxidase- and thiocyanate-dependent mechanism. Differentiated air-liquid interface cultures of rat tracheal epithelial cells provide a novel model to study airway epithelium-influenza interactions. The dual oxidase/lactoperoxidase/thiocyanate extracellular oxidative system producing hypothiocyanite is a fast and potent anti-influenza mechanism inactivating H1N2 viruses prior to infection of the epithelium.

Hippo/Yap signaling controls epithelial progenitor cell proliferation and differentiation in the embryonic and adult lung

PubMed Central

Lange, Alexander W.; Sridharan, Anusha; Xu, Yan; Stripp, Barry R.; Perl, Anne-Karina; Whitsett, Jeffrey A.

2015-01-01

The Hippo/Yap pathway is a well-conserved signaling cascade that regulates cell proliferation and differentiation to control organ size and stem/progenitor cell behavior. Following airway injury, Yap was dynamically regulated in regenerating airway epithelial cells. To determine the role of Hippo signaling in the lung, the mammalian Hippo kinases, Mst1 and Mst2, were deleted in epithelial cells of the embryonic and mature mouse lung. Mst1/2 deletion in the fetal lung enhanced proliferation and inhibited sacculation and epithelial cell differentiation. The transcriptional inhibition of cell proliferation and activation of differentiation during normal perinatal lung maturation were inversely regulated following embryonic Mst1/2 deletion. Ablation of Mst1/2 from bronchiolar epithelial cells in the adult lung caused airway hyperplasia and altered differentiation. Inhibitory Yap phosphorylation was decreased and Yap nuclear localization and transcriptional targets were increased after Mst1/2 deletion, consistent with canonical Hippo/Yap signaling. YAP potentiated cell proliferation and inhibited differentiation of human bronchial epithelial cells in vitro. Loss of Mst1/2 and expression of YAP regulated transcriptional targets controlling cell proliferation and differentiation, including Ajuba LIM protein. Ajuba was required for the effects of YAP on cell proliferation in vitro. Hippo/Yap signaling regulates Ajuba and controls proliferation and differentiation of lung epithelial progenitor cells. PMID:25480985

Human fetal enterocytes in vitro: modulation of the phenotype by extracellular matrix.

PubMed Central

Sanderson, I R; Ezzell, R M; Kedinger, M; Erlanger, M; Xu, Z X; Pringault, E; Leon-Robine, S; Louvard, D; Walker, W A

1996-01-01

The differentiation of small intestinal epithelial cells may require stimulation by microenvironmental factors in vivo. In this study, the effects of mesenchymal and luminal elements in nonmalignant epithelia] cells isolated from the human fetus were studied in vitro. Enterocytes from the human fetus were cultured and microenvironmental factors were added in stages, each stage more closely approximating the microenvironment in vivo. Four stages were examined: epithelial cells derived on plastic from intestinal culture and grown as a cell clone, the same cells grown on connective tissue support, primary epithelial explants grown on fibroblasts with a laminin base, and primary epithelial explants grown on fibroblasts and laminin with n-butyrate added to the incubation medium. The epithelial cell clone dedifferentiated when grown on plastic; however, the cells expressed cytokeratins and villin as evidence of their epithelial cell origin. Human connective tissue matrix from Engelbreth-Holm-Swarm sarcoma cells (Matrigel) modulated their phenotype: alkaline phosphatase activity increased, microvilli developed on their apical surface, and the profile of insulin-like growth factor binding proteins resembled that secreted by differentiated enterocytes. Epithelial cells taken directly from the human fetus as primary cultures and grown as explants on fibroblasts and laminin expressed greater specific enzyme activities in brush border membrane fractions than the cell clone. These activities were enhanced by the luminal molecule sodium butyrate. Thus the sequential addition of connective tissue and luminal molecules to nonmalignant epithelia] cells in vitro induces a spectrum of changes in the epithelial cell phenotype toward full differentiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8755542

Remodeling of the abdominal epithelial monolayer during the larva-pupa-adult transformation of Manduca.

PubMed

Nardi, James B; Bee, Charles Mark; Wallace, Catherine Lee

2018-06-01

During metamorphosis of insect epithelial monolayers, cells die, divide, and rearrange. In Drosophila undifferentiated diploid cells destined to form the adult cuticle of each abdominal segment segregate early in development from the surrounding polyploid larval epithelial cells of that segment as eight groups of diploid histoblast cells. The larval polyploid cells are programmed to die and be replaced by divisions and rearrangements of histoblast cells. By contrast, abdominal epithelial cells of Manduca larvae form a monolayer of cells representing different ploidy levels with no definitive segregation of diploid cells destined to form adult structures. These epithelial cells of mixed ploidy levels produce a thick smooth larval cuticle with sparsely distributed sensory bristles. Adult descendants of this larval monolayer produce a thinner cuticle with densely packed scale cells. The transition between these differentiated states of Manduca involves divisions of cells, changes in ploidy levels, and sorting of certain polyploid cells into circular rosette patches to minimize contacts of these polyploid cells with surrounding cells of equal or smaller size. Cells within the rosettes and some surrounding cells are destined to die and be replaced by remaining epithelial cells of uniform size and ploidy at pupa-adult apolysis. Copyright © 2018 Elsevier Inc. All rights reserved.

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

PubMed

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

2018-04-24

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

Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement

PubMed Central

Sorce, Barbara; Escobedo, Carlos; Toyoda, Yusuke; Stewart, Martin P.; Cattin, Cedric J.; Newton, Richard; Banerjee, Indranil; Stettler, Alexander; Roska, Botond; Eaton, Suzanne; Hyman, Anthony A.; Hierlemann, Andreas; Müller, Daniel J.

2015-01-01

Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells. PMID:26602832

Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement

NASA Astrophysics Data System (ADS)

Sorce, Barbara; Escobedo, Carlos; Toyoda, Yusuke; Stewart, Martin P.; Cattin, Cedric J.; Newton, Richard; Banerjee, Indranil; Stettler, Alexander; Roska, Botond; Eaton, Suzanne; Hyman, Anthony A.; Hierlemann, Andreas; Müller, Daniel J.

2015-11-01

Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells.

5-Oxo-ETE from Nasal Epithelial Cells Upregulates Eosinophil Cation Protein by Eosinophils in Nasal Polyps in vitro.

PubMed

Lin, Lin; Chen, Zheng; Tang, Xinyue; Dai, Fei; Wei, Jinjin; Sun, Guangbin

2018-06-13

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent eosinophil chemoattractant and activator that is synthesized not only in inflammatory cells but also in bronchial epithelial cells. The purpose of this study is to clarify whether 5-oxo-ETE can promote the production of eosinophil cation protein (ECP) by eosinophils in nasal polyps (NP) in vitro, and whether normal nasal epithelial cells can produce this lipid mediator in response to oxidative stress. Nasal biopsy samples were obtained from normal subjects or subjects with chronic rhinosinusitis with NP. The infiltration of eosinophil in NP was detected and cultured. After that, concentrations of ECP in eosinophil and NP cultures were evaluated after the treatment of 5-oxo-ETE or 5-oxo-ETE + its receptor (OXER) antagonist, pertussis toxin (PT). Then we studied the synthesis of 5-oxo-ETE after H2O2 stimulation by normal nasal epithelial cells and by epithelial cells of NP alone in the cultures, and also determined the OXER expression in NP. The number of infiltrative eosinophils in NP was increased. The ECP levels in eosinophil and NP cultures were enhanced after the administration of 5-oxo-ETE, and decreased by the PT treatment. 5-Oxo-ETE was upregulated in the cultures of nasal epithelial cells in the presence of H2O2 and of NP epithelial cells alone. The OXER was expressed in inflammatory cells, and not in epithelial cells. 5-Oxo-ETE produced by nasal epithelial cells may play a role in the formation and development of NP. © 2018 S. Karger AG, Basel.

Multi-functionality and plasticity characterize epithelial cells in Hydra

PubMed Central

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

2015-01-01

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

Apoptosis resistance in epithelial tumors is mediated by tumor-cell-derived interleukin-4.

PubMed

Todaro, M; Lombardo, Y; Francipane, M G; Alea, M Perez; Cammareri, P; Iovino, F; Di Stefano, A B; Di Bernardo, C; Agrusa, A; Condorelli, G; Walczak, H; Stassi, G

2008-04-01

We investigated the mechanisms involved in the resistance to cell death observed in epithelial cancers. Here, we identify that primary epithelial cancer cells from colon, breast and lung carcinomas express high levels of the antiapoptotic proteins PED, cFLIP, Bcl-xL and Bcl-2. These cancer cells produced interleukin-4 (IL-4), which amplified the expression levels of these antiapoptotic proteins and prevented cell death induced upon exposure to TRAIL or other drug agents. IL-4 blockade resulted in a significant decrease in the growth rate of epithelial cancer cells and sensitized them, both in vitro and in vivo, to apoptosis induction by TRAIL and chemotherapy via downregulation of the antiapoptotic factors PED, cFLIP, Bcl-xL and Bcl-2. Furthermore, we provide evidence that exogenous IL-4 was able to upregulate the expression levels of these antiapoptotic proteins and potently stabilized the growth of normal epithelial cells rendering them apoptosis resistant. In conclusion, IL-4 acts as an autocrine survival factor in epithelial cells. Our results indicate that inhibition of IL-4/IL-4R signaling may serve as a novel treatment for epithelial cancers.

Bioactive interleukin-1alpha is cytolytically released from Candida albicans-infected oral epithelial cells.

PubMed

Dongari-Bagtzoglou, A; Kashleva, H; Villar, C Cunha

2004-12-01

Oral epithelial cells are primary targets of Candida albicans in the oropharynx and may regulate the inflammatory host response to this pathogen. This investigation studied the mechanisms underlying interleukin-1alpha (IL-1alpha) release by oral epithelial cells and the role of IL-1alpha in regulating the mucosal inflammatory response to C. albicans. Infected oral epithelial cells released processed IL-1alpha protein in culture supernatants. The IL-1alpha generated was stored intracellularly and was released upon cell lysis. This was further supported by the fact that different C. albicans strains induced variable IL-1alpha release, depending on their cytolytic activity. IL-1alpha from C. albicans-infected oral epithelial cells upregulated proinflammatory cytokine secretion (IL-8 and GM-CSF) in uninfected oral epithelial or stromal cells. Our studies suggest that production of IL-1alpha, IL-8 and GM-CSF may take place in the oral mucosa in response to lytic infection of epithelial cells with C. albicans. This process can act as an early innate immune surveillance system and may contribute to the clinicopathologic signs of infection in the oral mucosa.

Phenotypic characterization of collagen gel embedded primary human breast epithelial cells in athymic nude mice.

PubMed

Yang, J; Guzman, R C; Popnikolov, N; Bandyopadhyay, G K; Christov, K; Collins, G; Nandi, S

1994-06-30

We have developed a method to characterize the phenotypes and tumorigenicity of dissociated human breast epithelial cells. The dissociated cells were first embedded in collagen gels and subsequently transplanted subcutaneously in vivo in athymic nude mice. The transplantation of dissociated epithelial cells from reduction mammoplasties, presumed to be normal, always resulted in normal histomorphology. Epithelial cells were arranged as short tubular structures consisting of lumina surrounded by epithelial cells with an occasional more complex branching structure. These outgrowths were surrounded by intact basement membrane and were embedded in collagen gel that, at termination, contained collagenous stroma with fibroblasts and blood vessels. In contrast, transplantation of dissociated breast epithelial cells from breast cancer specimens resulted in outgrowths with an invasive pattern infiltrating the collagen gel as well as frank invasion into vascular space, nerves and muscles. These observations were made long before the subsequent palpable stage which resulted if left in the mouse for a long enough time. The dissociated human breast epithelial cells thus retained their intrinsic property to undergo morphogenesis to reflect their original phenotype when placed in a suitable environment, the collagen gel.

Solitary chemoreceptor cells in the nasal cavity serve as sentinels of respiration

PubMed Central

Finger, Thomas E.; Böttger, Bärbel; Hansen, Anne; Anderson, Karl T.; Alimohammadi, Hessamedin; Silver, Wayne L.

2003-01-01

Inhalation of irritating substances leads to activation of the trigeminal nerve, triggering protective reflexes that include apnea or sneezing. Receptors for trigeminal irritants are generally assumed to be located exclusively on free nerve endings within the nasal epithelium, requiring that trigeminal irritants diffuse through the junctional barrier at the epithelial surface to activate receptors. We find, in both rats and mice, an extensive population of chemosensory cells that reach the surface of the nasal epithelium and form synaptic contacts with trigeminal afferent nerve fibers. These chemosensory cells express T2R “bitter-taste” receptors and α-gustducin, a G protein involved in chemosensory transduction. Functional studies indicate that bitter substances applied to the nasal epithelium activate the trigeminal nerve and evoke changes in respiratory rate. By extending to the surface of the nasal epithelium, these chemosensory cells serve to expand the repertoire of compounds that can activate trigeminal protective reflexes. The trigeminal chemoreceptor cells are likely to be remnants of the phylogenetically ancient population of solitary chemoreceptor cells found in the epithelium of all anamniote aquatic vertebrates. PMID:12857948

Solitary chemoreceptor cells in the nasal cavity serve as sentinels of respiration.

PubMed

Finger, Thomas E; Böttger, Bärbel; Hansen, Anne; Anderson, Karl T; Alimohammadi, Hessamedin; Silver, Wayne L

2003-07-22

Inhalation of irritating substances leads to activation of the trigeminal nerve, triggering protective reflexes that include apnea or sneezing. Receptors for trigeminal irritants are generally assumed to be located exclusively on free nerve endings within the nasal epithelium, requiring that trigeminal irritants diffuse through the junctional barrier at the epithelial surface to activate receptors. We find, in both rats and mice, an extensive population of chemosensory cells that reach the surface of the nasal epithelium and form synaptic contacts with trigeminal afferent nerve fibers. These chemosensory cells express T2R "bitter-taste" receptors and alpha-gustducin, a G protein involved in chemosensory transduction. Functional studies indicate that bitter substances applied to the nasal epithelium activate the trigeminal nerve and evoke changes in respiratory rate. By extending to the surface of the nasal epithelium, these chemosensory cells serve to expand the repertoire of compounds that can activate trigeminal protective reflexes. The trigeminal chemoreceptor cells are likely to be remnants of the phylogenetically ancient population of solitary chemoreceptor cells found in the epithelium of all anamniote aquatic vertebrates.

p16(INK4a) -mediated suppression of telomerase in normal and malignant human breast cells.

PubMed

Bazarov, Alexey V; Van Sluis, Marjolein; Hines, William C; Bassett, Ekaterina; Beliveau, Alain; Campeau, Eric; Mukhopadhyay, Rituparna; Lee, Won Jae; Melodyev, Sonya; Zaslavsky, Yuri; Lee, Leonard; Rodier, Francis; Chicas, Agustin; Lowe, Scott W; Benhattar, Jean; Ren, Bing; Campisi, Judith; Yaswen, Paul

2010-10-01

The cyclin-dependent kinase inhibitor p16(INK4a) (CDKN2A) is an important tumor suppressor gene frequently inactivated in human tumors. p16 suppresses the development of cancer by triggering an irreversible arrest of cell proliferation termed cellular senescence. Here, we describe another anti-oncogenic function of p16 in addition to its ability to halt cell cycle progression. We show that transient expression of p16 stably represses the hTERT gene, encoding the catalytic subunit of telomerase, in both normal and malignant breast epithelial cells. Short-term p16 expression increases the amount of histone H3 trimethylated on lysine 27 (H3K27) bound to the hTERT promoter, resulting in transcriptional silencing, likely mediated by polycomb complexes. Our results indicate that transient p16 exposure may prevent malignant progression in dividing cells by irreversible repression of genes, such as hTERT, whose activity is necessary for extensive self-renewal. © 2010 The Authors Aging Cell © 2010 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.

Hair Follicle Generation by Injections of Adult Human Follicular Epithelial and Dermal Papilla Cells into Nude Mice

PubMed Central

Nilforoushzadeh, Mohammadali; Rahimi Jameh, Elham; Jaffary, Fariba; Abolhasani, Ehsan; Keshtmand, Gelavizh; Zarkob, Hajar; Mohammadi, Parvaneh; Aghdami, Nasser

2017-01-01

Objective Dermal papilla and hair epithelial stem cells regulate hair formation and the growth cycle. Damage to or loss of these cells can cause hair loss. Although several studies claim to reconstitute hairs using rodent cells in an animal model, additional research is needed to develop a stable human hair follicle reconstitution protocol. In this study, we have evaluated hair induction by injecting adult cultured human dermal papilla cells and a mixture of hair epithelial and dermal papilla cells in a mouse model. Materials and Methods In this experimental study, discarded human scalp skins were used to obtain dermal papilla and hair epithelial cells. After separation, cells were cultured and assessed for their characteristics. We randomly allocated 15 C57BL/6 nude mice into three groups that received injections in their dorsal skin. The first group received cultured dermal papilla cells, the second group received a mixture of cultured epithelial and dermal papilla cells, and the third group (control) received a placebo [phosphate-buffered saline (PBS-)]. Results Histopathologic examination of the injection sites showed evidence of hair growth in samples that received cells compared with the control group. However, the group that received epithelial and dermal papilla cells had visible evidence of hair growth. PKH tracing confirmed the presence of transplanted cells in the new hair. Conclusion Our data showed that injection of a combination of adult human cultured dermal papilla and epithelial cells could induce hair growth in nude mice. This study emphasized that the combination of human adult cultured dermal papilla and epithelial cells could induce new hair in nude mice. PMID:28670518

In vitro studies with renal proximal tubule cells show direct cytotoxicity of Androctonus australis hector scorpion venom triggered by oxidative stress, caspase activation and apoptosis

PubMed Central

Saidani, Chanez; Hammoudi-Triki, Djelila; Laraba-Djebari, Fatima; Taub, Mary

2016-01-01

Scorpion envenomation injures a number of organs, including the kidney. Mechanisms proposed to explain the renal tubule injury include direct effects of venom on tubule epithelial cells, as well as indirect effects of the autonomic nervous system, and inflammation. Here, we report direct effects of Androctonus australis hector (Aah) scorpion venom on the viability of Renal Proximal Tubule (RPT) cells in vitro, unlike distal tubule and collecting duct cells. Extensive NucGreen nuclear staining was observed in immortalized rabbit RPT cells following treatment with Aah venom, consistent with cytotoxicity. The involvement of oxidative stress is supported by the observations that 1) anti-oxidants mitigated the Aah venom-induced decrease in the number of viable RPT cells, and 2) Aah venom-treated RPT cells were intensively stained with the CellROX® Deep Red reagent, an indicator of Reactive Oxygen Species (ROS). Relevance to normal RPT cells is supported by the red fluorescence observed in Aah venom treated primary rabbit RPT cell cultures following their incubation with the Flica reagent (indicative of caspase activation and apoptosis), and the green fluorescence of Sytox Green (indicative of dead cells). PMID:27470530

Role of contact inhibition of locomotion and junctional mechanics in epithelial collective responses to injury

NASA Astrophysics Data System (ADS)

Coburn, Luke; Lopez, Hender; Schouwenaar, Irin-Maya; Yap, Alpha S.; Lobaskin, Vladimir; Gomez, Guillermo A.

2018-03-01

Epithelial tissues form physically integrated barriers against the external environment protecting organs from infection and invasion. Within each tissue, epithelial cells respond to different challenges that can potentially compromise tissue integrity. In particular, cells collectively respond to injuries by reorganizing their cell-cell junctions and migrating directionally towards the sites of damage. Notwithstanding, the mechanisms that drive collective responses in epithelial aggregates remain poorly understood. In this work, we develop a minimal mechanistic model that is able to capture the essential features of epithelial collective responses to injuries. We show that a model that integrates the mechanics of cells at the cell-cell and cell-substrate interfaces as well as contact inhibition of locomotion (CIL) correctly predicts two key properties of epithelial response to injury as: (1) local relaxation of the tissue and (2) collective reorganization involving the extension of cryptic lamellipodia that extend, on average, up to 3 cell diameters from the site of injury and morphometric changes in the basal regions. Our model also suggests that active responses (like the actomyosin purse string and softening of cell-cell junctions) are needed to drive morphometric changes in the apical region. Therefore, our results highlight the importance of the crosstalk between junctional biomechanics, cell substrate adhesion, and CIL, as well as active responses, in guiding the collective rearrangements that are required to preserve the epithelial barrier in response to injury.

Gap Junction Protein Connexin 43 Serves as a Negative Marker for a Stem Cell-Containing Population of Human Limbal Epithelial Cells

PubMed Central

Chen, Zhuo; Evans, W. Howard; Pflugfelder, Stephen C.; Li, De-Quan

2010-01-01

This study evaluated whether the gap junction protein connexin (Cx) 43 could serve as a negative cell surface marker for human corneal epithelial stem cells. Cx43 expression was evaluated in corneo-limbal tissue and primary limbal epithelial cultures. Immunofluorescent staining and laser scanning confocal microscopy showed that Cx43 was strongly expressed in the corneal and limbal suprabasal epithelial cells, but the basal cells of the limbal epithelium were negative. Cx43 antibody stained mainly large cells but not small cells in primary limbal epithelial cultures. As determined by semiquantitative reverse transcription polymerase chain reaction (PCR) and real-time PCR, Cx43 mRNA was more abundant in the corneal than limbal epithelia, and it was expressed in higher levels in mature limbal epithelial cultures. Using GAP11, a rabbit polyclonal antibody against the Cx32 extracellular loop 2 (151–187), a sequence that is highly homologous in Cx43, the Cx43dim and Cx43bright cells were selected from primary limbal epithelial cultures by fluorescence-activated cell sorting and were evaluated for stem cell properties. These Cx43dim and Cx43bright cells were confirmed by their expression levels of Cx43 protein and mRNA. The Cx43dim cells were found to contain higher percentages of slow-cycling bromodeoxyuridine (BrdU)-label retaining cells and the cells that were positive for stem cell-associated markers p63, ABCG2, and integrin β1 and negative for differentiation markers K3 and involucrin. The Cx43dim cells possessed a greater proliferative potential than Cx43bright cells and nonfractionated cells as evaluated by BrdU incorporation, colony-forming efficiency, and growth capacity. Our findings suggest that human limbal basal cells do not express connexin 43, which could serve as a negative cell surface marker for the stem cell-containing population of human limbal epithelial cells. PMID:16424398

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

PubMed

Hara, Yusuke

2017-06-01

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

Epithelial self-healing is recapitulated by a 3D biomimetic E-cadherin junction.

PubMed

Cohen, Daniel J; Gloerich, Martijn; Nelson, W James

2016-12-20

Epithelial monolayers undergo self-healing when wounded. During healing, cells collectively migrate into the wound site, and the converging tissue fronts collide and form a stable interface. To heal, migrating tissues must form cell-cell adhesions and reorganize from the front-rear polarity characteristic of cell migration to the apical-basal polarity of an epithelium. However, identifying the "stop signal" that induces colliding tissues to cease migrating and heal remains an open question. Epithelial cells form integrin-based adhesions to the basal extracellular matrix (ECM) and E-cadherin-mediated cell-cell adhesions on the orthogonal, lateral surfaces between cells. Current biological tools have been unable to probe this multicellular 3D interface to determine the stop signal. We addressed this problem by developing a unique biointerface that mimicked the 3D organization of epithelial cell adhesions. This "minimal tissue mimic" (MTM) comprised a basal ECM substrate and a vertical surface coated with purified extracellular domain of E-cadherin, and was designed for collision with the healing edge of an epithelial monolayer. Three-dimensional imaging showed that adhesions formed between cells, and the E-cadherin-coated MTM resembled the morphology and dynamics of native epithelial cell-cell junctions and induced the same polarity transition that occurs during epithelial self-healing. These results indicate that E-cadherin presented in the proper 3D context constitutes a minimum essential stop signal to induce self-healing. That the Ecad:Fc MTM stably integrated into an epithelial tissue and reduced migration at the interface suggests that this biointerface is a complimentary approach to existing tissue-material interfaces.

Establishment of a Human Conjunctival Epithelial Cell Line Lacking the Functional Tacstd2 Gene (An American Ophthalmological Society Thesis)

PubMed Central

Kinoshita, Shigeru; Kawasaki, Satoshi; Kitazawa, Koji; Shinomiya, Katsuhiko

2012-01-01

Purpose: To report the establishment of a human conjunctival epithelial cell line lacking the functional tumor-associated calcium signal transducer 2 (TACSTD2) gene to be used as an in vitro model of gelatinous drop-like corneal dystrophy (GDLD), a rare disease in which the corneal epithelial barrier function is significantly compromized by the loss of function mutation of the TACSTD2 gene. Methods: A small piece of conjunctival tissue was obtained from a GDLD patient. The conjunctival epithelial cells were enzymatically separated and dissociated from the tissue and immortalized by the lentiviral introduction of the SV40 large T antigen and human telomerase reverse transcriptase (hTERT) genes. Population doubling, protein expression, and transepithelial resistance (TER) analyses were performed to assess the appropriateness of the established cell line as an in vitro model for GDLD. Results: The life span of the established cell line was found to be significantly elongated compared to nontransfected conjunctival epithelial cells. The SV40 large T antigen and hTERT genes were stably expressed in the established cell line. The protein expression level of the tight junction–related proteins was significantly low compared to the immortalized normal conjunctival epithelial cell line. TER of the established cell line was found to be significantly low compared to the immortalized normal conjunctival epithelial cell line. Conclusions: Our conjunctival epithelial cell line was successfully immortalized and well mimicked several features of GDLD corneas. This cell line may be useful for the elucidation of the pathogenesis of GDLD and for the development of novel treatments for GDLD. PMID:23818740

Eosinophils Promote Epithelial to Mesenchymal Transition of Bronchial Epithelial Cells

PubMed Central

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

2013-01-01

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

Niche induced cell death and epithelial phagocytosis regulate hair follicle stem cell pool

PubMed Central

Mesa, Kailin R.; Rompolas, Panteleimon; Zito, Giovanni; Myung, Peggy; Sun, Thomas Yang; Brown, Samara; Gonzalez, David; Blagoev, Krastan B.; Haberman, Ann M.; Greco, Valentina

2015-01-01

Summary Tissue homeostasis is achieved through a balance of cell production (growth) and elimination (regression)1,2. Contrary to tissue growth, the cells and molecular signals required for tissue regression remain unknown. To investigate physiological tissue regression, we use the mouse hair follicle, which cycles stereotypically between phases of growth and regression while maintaining a pool of stem cells to perpetuate tissue regeneration3. Here we show by intravital microscopy in live mice4–6 that the regression phase eliminates the majority of the epithelial cells by two distinct mechanisms: terminal differentiation of suprabasal cells and a spatial gradient of apoptosis of basal cells. Furthermore, we demonstrate that basal epithelial cells collectively act as phagocytes to clear dying epithelial neighbors. Through cellular and genetic ablation we show that epithelial cell death is extrinsically induced through TGFβ activation and mesenchymal crosstalk. Strikingly, our data show that regression acts to reduce the stem cell pool as inhibition of regression results in excess basal epithelial cells with regenerative abilities. This study identifies the cellular behaviors and molecular mechanisms of regression that counterbalance growth to maintain tissue homeostasis. PMID:25849774

The farnesyltransferase inhibitor, LB42708, inhibits growth and induces apoptosis irreversibly in H-ras and K-ras-transformed rat intestinal epithelial cells

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

Kim, Han-Soo; Kim, Ju Won; Gang, Jingu

2006-09-15

LB42708 (LB7) and LB42908 (LB9) are pyrrole-based orally active farnesyltransferase inhibitors (FTIs) that have similar structures. The in vitro potencies of these compounds against FTase and GGTase I are remarkably similar, and yet they display different activity in apoptosis induction and morphological reversion of ras-transformed rat intestinal epithelial (RIE) cells. Both FTIs induced cell death despite K-ras prenylation, implying the participation of Ras-independent mechanism(s). Growth inhibition by these two FTIs was accompanied by G1 and G2/M cell cycle arrests in H-ras and K-ras-transformed RIE cells, respectively. We identified three key markers, p21{sup CIP1/WAF1}, RhoB and EGFR, that can explain themore » differences in the molecular mechanism of action between two FTIs. Only LB7 induced the upregulation of p21{sup CIP1/WAF1} and RhoB above the basal level that led to the cell cycle arrest and to distinct morphological alterations of ras-transformed RIE cells. Both FTIs successfully inhibited the ERK and activated JNK in RIE/K-ras cells. While the addition of conditioned medium from RIE/K-ras reversed the growth inhibition of ras-transformed RIE cells by LB9, it failed to overcome the growth inhibitory effect of LB7 in both H-ras- and K-ras-transformed RIE cells. We found that LB7, but not LB9, decreased the expression of EGFRs that confers the cellular unresponsiveness to EGFR ligands. These results suggest that LB7 causes the induction of p21{sup CIP1/WAF1} and RhoB and downregulation of EGFR that may serve as critical steps in the mechanism by which FTIs trigger irreversible inhibitions on the cell growth and apoptosis in ras-transformed cells.« less

Activation of Neurokinin-1 Receptors during Ozone Inhalation Contributes to Epithelial Injury and Repair

PubMed Central

Oslund, Karen L.; Hyde, Dallas M.; Putney, Leialoha F.; Alfaro, Mario F.; Walby, William F.; Tyler, Nancy K.; Schelegle, Edward S.

2008-01-01

We investigated the importance of neurokinin (NK)-1 receptors in epithelial injury and repair and neutrophil function. Conscious Wistar rats were exposed to 1 ppm ozone or filtered air for 8 hours, followed by an 8-hour postexposure period. Before exposure, we administered either the NK-1 receptor antagonist, SR140333, or saline as a control. Ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, whole mounts of airway dissected lung lobes were immunostained for 5-bromo-2′-deoxyuridine, a marker of epithelial proliferation. Both ethidium homodimer and 5-bromo-2′-deoxyuridine-positive epithelial cells were quantified in specific airway generations. Rats treated with the NK-1 receptor antagonist had significantly reduced epithelial injury and epithelial proliferation compared with control rats. Sections of terminal bronchioles showed no significant difference in the number of neutrophils in airways between groups. In addition, staining ozone-exposed lung sections for active caspase 3 showed no apoptotic cells, but ethidium-positive cells colocalized with the orphan nuclear receptor, Nur77, a marker of nonapoptotic, programmed cell death mediated by the NK-1 receptor. An immortalized human airway epithelial cell line, human bronchial epithelial-1, showed no significant difference in the number of oxidant stress–positive cells during exposure to hydrogen peroxide and a range of SR140333 doses, demonstrating no antioxidant effect of the receptor antagonist. We conclude that activation of the NK-1 receptor during acute ozone inhalation contributes to epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways. PMID:18390473

Aryl hydrocarbon receptor-dependent up-regulation of the heterodimeric amino acid transporter LAT1 (SLC7A5)/CD98hc (SLC3A2) by diesel exhaust particle extract in human bronchial epithelial cells

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

Le Vee, Marc; Jouan, Elodie; Lecureur, Valérie

The heterodimeric L-type amino acid transporter (LAT) 1/CD98hc is overexpressed in lung cancers with a poor prognosis factor. Factors that contribute to LAT1/CD98hc overexpression in lung cells remain however to be determined, but the implication of atmospheric pollution can be suspected. The present study was therefore designed to analyze the effects of diesel exhaust particle (DEP) extract (DEPe) on LAT1/CD98hc expression in bronchial epithelial BEAS-2B cells. Exposure to DEPe up-regulated LAT1 and CD98hc mRNA levels in a concentration-dependent manner, with DEPe EC{sub 50} values (around 0.2 μg/mL) relevant to environmental situations. DEPe concomitantly induced LAT1/CD98hc protein expression and LAT1-mediated leucinemore » accumulation in BEAS-2B cells. Inhibition of the aryl hydrocarbon receptor (AhR) pathway through the use of a chemical AhR antagonist or the siRNA-mediated silencing of AhR expression was next found to prevent DEPe-mediated induction of LAT1/CD98hc, indicating that this regulation depends on AhR, known to be activated by major chemical DEP components like polycyclic aromatic hydrocarbons. DEPe exposure was finally shown to induce mRNA expression and activity of matrix metalloproteinase (MMP)-2 in BEAS-2B cells, in a CD98hc/focal adhesion kinase (FAK)/extracellular regulated kinase (ERK) manner, thus suggesting that DEPe-mediated induction of CD98hc triggers activation of the integrin/FAK/ERK signaling pathway known to be involved in MMP-2 regulation. Taken together, these data demonstrate that exposure to DEPe induces functional overexpression of the amino acid transporter LAT1/CD98hc in lung cells. Such a regulation may participate to pulmonary carcinogenic effects of DEPs, owing to the well-documented contribution of LAT1 and CD98hc to cancer development. - Highlights: • The amino acid transporter LAT1/CD98hc is up-regulated in DEPe-treated lung cells. • The aryl hydrocarbon receptor is involved in DEPe-triggered induction of LAT1/CD98hc. • DEPe enhances LAT1-mediated accumulation of leucine in lung cells. • DEPe increases MMP-2 expression and activity in a CD98hc/FAK/ERK-dependent manner.« less

Ductal cancers of the pancreas frequently express markers of gastrointestinal epithelial cells.

PubMed

Sessa, F; Bonato, M; Frigerio, B; Capella, C; Solcia, E; Prat, M; Bara, J; Samloff, I M

1990-06-01

It has been found by immunohistochemical staining that antigens normally found in gastric and/or intestinal epithelial cells are expressed in most differentiated duct cell carcinomas of the pancreas. Among 88 such tumors, 93% and 92%, respectively, expressed M1 and cathepsin E, markers of gastric surface-foveolar epithelial cells, 51% expressed pepsinogen II, a marker of gastroduodenal mucopeptic cells, 48% expressed CAR-5, a marker of colorectal epithelial cells, and 35% expressed M3SI, a marker of small intestinal goblet cells. Most of the tumors also expressed normal pancreatic duct antigens; 97% expressed DU-PAN-2, and 59% expressed N-terminus gastrin-releasing peptide. In agreement with these findings, electron microscopy revealed malignant cells with fine structural features of gastric foveolar cells, gastric mucopeptic cells, intestinal goblet cells, intestinal columnar cells, pancreatic duct epithelial cells, and cells with features of more than one cell type. Normal pancreatic duct epithelium did not express any marker of gastrointestinal epithelial cells, whereas such benign lesions as mucinous cell hypertrophy and papillary hyperplasia commonly expressed gut-type antigens but rarely expressed pancreatic duct cell markers. By contrast, lesions characterized by atypical papillary hyperplasia commonly expressed both gastric and pancreatic duct cell markers. Metaplastic pyloric-type glands expressed pepsinogen II and, except for their expression of cathepsin E, were indistinguishable from normal pyloric glands. In marked contrast, the immunohistochemical and ultrastructural features of 14 ductuloacinar cell tumors were those of cells lining terminal ductules, centroacinar cells, and/or acinar cells; none expressed any gut-type antigen. The results indicate that gastrointestinal differentiation is common in both benign and malignant lesions of pancreatic duct epithelium and suggest that duct cell carcinomas are histogenetically related to gastric- and intestinal-type metaplastic changes of epithelial cells lining the main and interlobular ducts of the pancreas.

Pathological changes of thymic epithelial cells and autoimmune disease in NZB, NZW and (NZB × NZW)F1 mice

PubMed Central

Vries, M. J. De; Hijmans, W.

1967-01-01

An extensive histological study was carried out of NZB, NZW and (NZB × NZW)F1, (BWF1), mice of all ages between birth and 18 months. The thymuses of these mice were compared to those of three normal mouse strains. The study of the NZW mice showed that these mice, although they only occasionally have weakly positive Coombs' tests, may develop a renal disease probably of an autoimmune nature, similar to that of the NZB and the BWF1 mice. Mice of all the three NZ strains developed lesions of the skin, liver, intestines, lymphatic tissues and kidneys much resembling those occurring in human systemic lupus erythematosus (SLE), neonatally thymectomized mice and, with the exception of the renal changes, the lesions of graft versus host disease. The comparative study of the thymus in autoimmune and normal strains, revealed that important changes of the large medullary epithelial cells, involved in the formation of Hassall's corpuscles, occur very early in the three autoimmune strains. In the NZB mice the large epithelial cells are severely decreased in number in the first weeks following birth. The depletion of epithelial cells could be ascribed to a secondary degeneration of these cells soon after birth. In contrast with the NZB mice, an extensive hyperplasia of the large epithelial cells and Hassall's corpuscles was observed in the NZW and BWF1 mice, and was already apparent in the newborn animal. Many of the epithelial aggregates seemed to have been invaded by lymphoid cells. Both epithelial cells and the lymphoid cells engaged in this process showed a variety of degenerative changes. As in the NZB, a depletion of epithelial cells occurred in a later phase, at the age of 8 months in the BWF1 and at 1 year in the NZW. In the majority of young mice of the normal strains invasion of islands of epithelial cells by lymphoid cells may also be observed, although this process is far less extensive than in the autoimmune strains and does not result in either epithelial hyperplasia or depletion of epithelial cells. The described phenomenon of invasion of epithelial structures in the thymus by subsequently disintegrating lymphoid cells seems to support Burnet's concept, that so-called `forbidden clones' of lymphoid cells are eliminated in the thymus. ImagesFIG. 18FIG. 19FIG. 20FIG. 2FIG. 3FIG. 4FIG. 5FIG. 6FIG. 7FIG. 8FIG. 9FIG. 10FIG. 11FIG. 12FIG. 13FIG. 14FIG. 15FIG. 16FIG. 17 PMID:6020121

Moderate plasma activated media suppresses proliferation and migration of MDCK epithelial cells

NASA Astrophysics Data System (ADS)

Mohades, Soheila; Laroussi, Mounir; Maruthamuthu, Venkat

2017-05-01

Low-temperature plasma has been shown to have diverse biomedical uses, including its applications in cancer and wound healing. One recent approach in treating mammalian cells with plasma is through the use of plasma activated media (PAM), which is produced by exposing cell culture media to plasma. While the adverse effects of PAM treatment on cancerous epithelial cell lines have been recently studied, much less is known about the interaction of PAM with normal epithelial cells. In this paper, non-cancerous canine kidney MDCK (Madin-Darby Canine Kidney) epithelial cells were treated by PAM and time-lapse microscopy was used to directly monitor their proliferation and random migration upon treatment. While longer durations of PAM treatment led to cell death, we found that moderate levels of PAM treatment inhibited proliferation in these epithelial cells. We also found that PAM treatment reduced random cell migration within epithelial islands. Immunofluorescence staining showed that while there were no major changes in the actin/adhesion apparatus, there was a significant change in the nuclear localization of proliferation marker Ki-67, consistent with our time-lapse results.

Coupling between apical tension and basal adhesion allow epithelia to collectively sense and respond to substrate topography over long distances.

PubMed

Broaders, Kyle E; Cerchiari, Alec E; Gartner, Zev J

2015-12-01

Epithelial sheets fold into complex topographies that contribute to their function in vivo. Cells can sense and respond to substrate topography in their immediate vicinity by modulating their interfacial mechanics, but the extent to which these mechanical properties contribute to their ability to sense substrate topography across length scales larger than a single cell has not been explored in detail. To study the relationship between the interfacial mechanics of single cells and their collective behavior as tissues, we grew cell-sheets on substrates engraved with surface features spanning macroscopic length-scales. We found that many epithelial cell-types sense and respond to substrate topography, even when it is locally nearly planar. Cells clear or detach from regions of local negative curvature, but not from regions with positive or no curvature. We investigated this phenomenon using a finite element model where substrate topography is coupled to epithelial response through a balance of tissue contractility and adhesive forces. The model correctly predicts the focal sites of cell-clearing and epithelial detachment. Furthermore, the model predicts that local tissue response to substrate curvature is a function of the surrounding topography of the substrate across long distances. Analysis of cell-cell and cell-substrate contact angles suggests a relationship between these single-cell interfacial properties, epithelial interfacial properties, and collective epithelial response to substrate topography. Finally, we show that contact angles change upon activation of oncogenes or inhibition of cell-contractility, and that these changes correlate with collective epithelial response. Our results demonstrate that in mechanically integrated epithelial sheets, cell contractility can be transmitted through multiple cells and focused by substrate topography to affect a behavioral response at distant sites.

Cationic polystyrene nanospheres induce autophagic cell death through the induction of endoplasmic reticulum stress

NASA Astrophysics Data System (ADS)

Chiu, Hui-Wen; Xia, Tian; Lee, Yu-Hsuan; Chen, Chun-Wan; Tsai, Jui-Chen; Wang, Ying-Jan

2014-12-01

Nanoparticles (NPs) have been used to produce a wide range of products that have applications in imaging and drug delivery in medicine. Due to their chemical stability, well-controlled sizes and surface charges, polystyrene (PS) NPs have been developed as biosensors and drug delivery carriers. However, the possible adverse biological effects and underlying mechanisms are still unclear. Recently, autophagy has been implicated in the regulation of cell death. In this study, we evaluated a library of PS NPs with different surface charges. We found that NH2-labeled polystyrene (NH2-PS) nanospheres were highly toxic with enhanced uptake in macrophage (RAW 264.7) and lung epithelial (BEAS-2B) cells. Furthermore, NH2-PS could induce autophagic cell death. NH2-PS increased autophagic flux due to reactive oxygen species (ROS) generation and endoplasmic reticulum (ER) stress caused by misfolded protein aggregation. The inhibition of ER stress decreased cytotoxicity and autophagy in the NH2-PS-treated cells. In addition, the Akt/mTOR and AMPK signaling pathways were involved in the regulation of NH2-PS-triggered autophagic cell death. These results suggest an important role of autophagy in cationic NP-induced cell death and provide mechanistic insights into the inhibition of the toxicity and safe material design.Nanoparticles (NPs) have been used to produce a wide range of products that have applications in imaging and drug delivery in medicine. Due to their chemical stability, well-controlled sizes and surface charges, polystyrene (PS) NPs have been developed as biosensors and drug delivery carriers. However, the possible adverse biological effects and underlying mechanisms are still unclear. Recently, autophagy has been implicated in the regulation of cell death. In this study, we evaluated a library of PS NPs with different surface charges. We found that NH2-labeled polystyrene (NH2-PS) nanospheres were highly toxic with enhanced uptake in macrophage (RAW 264.7) and lung epithelial (BEAS-2B) cells. Furthermore, NH2-PS could induce autophagic cell death. NH2-PS increased autophagic flux due to reactive oxygen species (ROS) generation and endoplasmic reticulum (ER) stress caused by misfolded protein aggregation. The inhibition of ER stress decreased cytotoxicity and autophagy in the NH2-PS-treated cells. In addition, the Akt/mTOR and AMPK signaling pathways were involved in the regulation of NH2-PS-triggered autophagic cell death. These results suggest an important role of autophagy in cationic NP-induced cell death and provide mechanistic insights into the inhibition of the toxicity and safe material design. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr05509h

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

PubMed

Gao, Yang; Li, Shu; Li, Qinglei

2014-08-01

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

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

PubMed Central

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

2016-01-01

Background The purpose of the study is to elicit the epigenetic mechanism involving CCCTC binding factor (CTCF)-mediated chromatin remodeling that regulates PAX6 gene interaction with differentiation-associated genes to control corneal epithelial differentiation. Methods Cell cycle progression and specific keratin expressions were measured to monitor changes of differentiation-induced primary human limbal stem/progenitor (HLS/P), human corneal epithelial (HCE) and human telomerase-immortalized corneal epithelial (HTCE) cells. PAX6-interactive and differentiation-associated genes in chromatin remodeling mediated by the epigenetic factor CTCF were detected by circular chromosome conformation capture (4C) and ChIP (Chromatin immunoprecipitation)-on-chip approaches, and verified by FISH (Fluorescent in situ hybridization). Furthermore, CTCF activities were altered by CTCF-shRNA to study the effect of CTCF on mediating interaction of Pax6 and differentiation-associated genes in corneal epithelial cell fate. Results Our results demonstrated that differentiation-induced human corneal epithelial cells expressed typical corneal epithelial characteristics including morphological changes, increased keratin12 expression and G0/G1 accumulations. Expressions of CTCF and PAX6 were suppressed and elevated following the process of differentiation, respectively. During corneal epithelial cell differentiation, differentiation-induced RCN1 and ADAM17 were found interacting with PAX6 in the process of CTCF-mediated chromatin remodeling detected by 4C and verified by ChIP-on-chip and FISH. Diminished CTCF mRNA with CTCF-shRNA in HTCE cells weakened the interaction of PAX6 gene in controlling RCN1/ADAM17 and enhanced early onset of the genes in cell differentiation. Conclusion Our results explain how epigenetic factor CTCF-mediated chromatin remodeling regulates interactions between eye-specific PAX6 and those genes that are induced/associated with cell differentiation to modulate corneal epithelial cell-specific differentiation. PMID:27583466

Rab5-regulated endocytosis plays a crucial role in apical extrusion of transformed cells.

PubMed

Saitoh, Sayaka; Maruyama, Takeshi; Yako, Yuta; Kajita, Mihoko; Fujioka, Yoichiro; Ohba, Yusuke; Kasai, Nobuhiro; Sugama, Natsu; Kon, Shunsuke; Ishikawa, Susumu; Hayashi, Takashi; Yamazaki, Tomohiro; Tada, Masazumi; Fujita, Yasuyuki

2017-03-21

Newly emerging transformed cells are often eliminated from epithelial tissues. Recent studies have revealed that this cancer-preventive process involves the interaction with the surrounding normal epithelial cells; however, the molecular mechanisms underlying this phenomenon remain largely unknown. In this study, using mammalian cell culture and zebrafish embryo systems, we have elucidated the functional involvement of endocytosis in the elimination of RasV12-transformed cells. First, we show that Rab5, a crucial regulator of endocytosis, is accumulated in RasV12-transformed cells that are surrounded by normal epithelial cells, which is accompanied by up-regulation of clathrin-dependent endocytosis. Addition of chlorpromazine or coexpression of a dominant-negative mutant of Rab5 suppresses apical extrusion of RasV12 cells from the epithelium. We also show in zebrafish embryos that Rab5 plays an important role in the elimination of transformed cells from the enveloping layer epithelium. In addition, Rab5-mediated endocytosis of E-cadherin is enhanced at the boundary between normal and RasV12 cells. Rab5 functions upstream of epithelial protein lost in neoplasm (EPLIN), which plays a positive role in apical extrusion of RasV12 cells by regulating protein kinase A. Furthermore, we have revealed that epithelial defense against cancer (EDAC) from normal epithelial cells substantially impacts on Rab5 accumulation in the neighboring transformed cells. This report demonstrates that Rab5-mediated endocytosis is a crucial regulator for the competitive interaction between normal and transformed epithelial cells in mammals.

Hippo/Yap signaling controls epithelial progenitor cell proliferation and differentiation in the embryonic and adult lung.

PubMed

Lange, Alexander W; Sridharan, Anusha; Xu, Yan; Stripp, Barry R; Perl, Anne-Karina; Whitsett, Jeffrey A

2015-02-01

The Hippo/Yap pathway is a well-conserved signaling cascade that regulates cell proliferation and differentiation to control organ size and stem/progenitor cell behavior. Following airway injury, Yap was dynamically regulated in regenerating airway epithelial cells. To determine the role of Hippo signaling in the lung, the mammalian Hippo kinases, Mst1 and Mst2, were deleted in epithelial cells of the embryonic and mature mouse lung. Mst1/2 deletion in the fetal lung enhanced proliferation and inhibited sacculation and epithelial cell differentiation. The transcriptional inhibition of cell proliferation and activation of differentiation during normal perinatal lung maturation were inversely regulated following embryonic Mst1/2 deletion. Ablation of Mst1/2 from bronchiolar epithelial cells in the adult lung caused airway hyperplasia and altered differentiation. Inhibitory Yap phosphorylation was decreased and Yap nuclear localization and transcriptional targets were increased after Mst1/2 deletion, consistent with canonical Hippo/Yap signaling. YAP potentiated cell proliferation and inhibited differentiation of human bronchial epithelial cells in vitro. Loss of Mst1/2 and expression of YAP regulated transcriptional targets controlling cell proliferation and differentiation, including Ajuba LIM protein. Ajuba was required for the effects of YAP on cell proliferation in vitro. Hippo/Yap signaling regulates Ajuba and controls proliferation and differentiation of lung epithelial progenitor cells. © The Author (2014). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

Herpes Simplex Virus Type 1 Neuronal Infection Perturbs Golgi Apparatus Integrity through Activation of Src Tyrosine Kinase and Dyn-2 GTPase

PubMed Central

Martin, Carolina; Leyton, Luis; Hott, Melissa; Arancibia, Yennyfer; Spichiger, Carlos; McNiven, Mark A.; Court, Felipe A.; Concha, Margarita I.; Burgos, Patricia V.; Otth, Carola

2017-01-01

Herpes simplex virus type 1 (HSV-1) is a ubiquitous pathogen that establishes a latent persistent neuronal infection in humans. The pathogenic effects of repeated viral reactivation in infected neurons are still unknown. Several studies have reported that during HSV-1 epithelial infection, the virus could modulate diverse cell signaling pathways remodeling the Golgi apparatus (GA) membranes, but the molecular mechanisms implicated, and the functional consequences to neurons is currently unknown. Here we report that infection of primary neuronal cultures with HSV-1 triggers Src tyrosine kinase activation and subsequent phosphorylation of Dynamin 2 GTPase, two players with a role in GA integrity maintenance. Immunofluorescence analyses showed that HSV-1 productive neuronal infection caused a scattered and fragmented distribution of the GA through the cytoplasm, contrasting with the uniform perinuclear distribution pattern observed in control cells. In addition, transmission electron microscopy revealed swollen cisternae and disorganized stacks in HSV-1 infected neurons compared to control cells. Interestingly, PP2, a selective inhibitor for Src-family kinases markedly reduced these morphological alterations of the GA induced by HSV-1 infection strongly supporting the possible involvement of Src tyrosine kinase. Finally, we showed that HSV-1 tegument protein VP11/12 is necessary but not sufficient to induce Dyn2 phosphorylation. Altogether, these results show that HSV-1 neuronal infection triggers activation of Src tyrosine kinase, phosphorylation of Dynamin 2 GTPase, and perturbation of GA integrity. These findings suggest a possible neuropathogenic mechanism triggered by HSV-1 infection, which could involve dysfunction of the secretory system in neurons and central nervous system. PMID:28879169

Herpes Simplex Virus Type 1 Neuronal Infection Perturbs Golgi Apparatus Integrity through Activation of Src Tyrosine Kinase and Dyn-2 GTPase.

PubMed

Martin, Carolina; Leyton, Luis; Hott, Melissa; Arancibia, Yennyfer; Spichiger, Carlos; McNiven, Mark A; Court, Felipe A; Concha, Margarita I; Burgos, Patricia V; Otth, Carola

2017-01-01

Herpes simplex virus type 1 (HSV-1) is a ubiquitous pathogen that establishes a latent persistent neuronal infection in humans. The pathogenic effects of repeated viral reactivation in infected neurons are still unknown. Several studies have reported that during HSV-1 epithelial infection, the virus could modulate diverse cell signaling pathways remodeling the Golgi apparatus (GA) membranes, but the molecular mechanisms implicated, and the functional consequences to neurons is currently unknown. Here we report that infection of primary neuronal cultures with HSV-1 triggers Src tyrosine kinase activation and subsequent phosphorylation of Dynamin 2 GTPase, two players with a role in GA integrity maintenance. Immunofluorescence analyses showed that HSV-1 productive neuronal infection caused a scattered and fragmented distribution of the GA through the cytoplasm, contrasting with the uniform perinuclear distribution pattern observed in control cells. In addition, transmission electron microscopy revealed swollen cisternae and disorganized stacks in HSV-1 infected neurons compared to control cells. Interestingly, PP2, a selective inhibitor for Src-family kinases markedly reduced these morphological alterations of the GA induced by HSV-1 infection strongly supporting the possible involvement of Src tyrosine kinase. Finally, we showed that HSV-1 tegument protein VP11/12 is necessary but not sufficient to induce Dyn2 phosphorylation. Altogether, these results show that HSV-1 neuronal infection triggers activation of Src tyrosine kinase, phosphorylation of Dynamin 2 GTPase, and perturbation of GA integrity. These findings suggest a possible neuropathogenic mechanism triggered by HSV-1 infection, which could involve dysfunction of the secretory system in neurons and central nervous system.