Epithelial Cell Clones Research Articles

Elevation of IL-8 secretion induced by PEDV infection via NF-κB signaling pathway.

Porcine epidemic diarrhea virus (PEDV) is associated with severe enteritis, which contributes to high mortality in piglets. The aim of this study was to describe molecular mechanisms associated with proinflammatory cytokine(s) production during PEDV infection. We showed that infection of porcine intestine epithelial cell clone J2 (IPEC-J2) with PEDV induces a gradual increase in interleukin 8 (IL-8) production at different time points, as well as infection of Vero E6 with PEDV. The secretion of IL-8 in these two cell lines infected with PEDV is related to the activation of NF-κB. Furthermore, the cells expressing PEDV M or E protein can induce the upregulation of IL-8. These findings suggest that the IL-8 production can be the initiator of inflammatory response by the host cells upon PEDV infection.

#2804 Mechanisms of cAMP-induced cyst formation observed in Pkhd1-KO cells

Abstract Background and Aims Loss of ciliary protein function leads to altered epithelial properties in hereditary polycystic kidney diseases. To address molecular aspects of defective epithelial homeostasis, monolayered epithelial spheroids can be used to analyze lack of protein function and consequences of pharmacological intervention. Here, we employed epithelial cell clones deficient for fibrocystin/polyductin (FPC) protein function, the origin of ARPKD, to study the impact of FPC cytoplasmic domain constructs on cAMP-induced cystogenesis. Method We used pl-MDCK, sub-cloned principal-like cell lines, with CRISPR/Cas9-based genetic knockout (KO) of Pkhd1 / FPC, and corresponding controls. Cells were grown in matrigel to allow formation of epithelial spheroids within 3 days. Forskolin (Fsk) treatment was employed to induce cAMP-mediated cyst growth mimicking disease conditions. Measurement of proportional lumen, the ratio of lumen to spheroid area, allowed detection the enhanced water and ion transport across the barrier as characteristic for secretory epithelia. Cystogenic signals were modulated by expression of the FPC C-terminal domain constructs and/or pharmacological inhibitors, and involvement of ion channels was addressed. Results In Pkhd1-KO cell lines, enhanced cAMP levels resulted in massive lumen expansion of epithelial spheroids with no increase in cell number. The rise in proportional lumen was sensitive to inhibition of chloride-channels, CFTR and TMEM16A, and Src kinase. Fsk-mediated cyst induction led to activation and increased Y705 phosphorylation of STAT3. To address the impact of FPC on cystogenic signaling, expression of a membrane-bound FPC C-terminal protein domain was studied, and its processing and intracellular localization determined. Controlled expression of the FPC cytoplasmic domain in Pkhd1-KO cell lines suppressed Fsk-induced cyst formation. In addition, the FPC domain reduced activation of Src and STAT3, leading to lower STAT3-dependent transcription. Conclusion In FPC-deficient pl-MDCK cells, cyst formation stimulated by high cAMP levels is associated with enhanced Src/STAT3 signaling. Expression of the FPC cytoplasmic domain, leading to a gain-of-function, suppresses Src and STAT3 activity in Pkhd1-KO epithelia and limits cyst growth in vitro. Control of cystogenic Src/STAT3 signaling can be considered a physiologic function of the FPC protein.

Metabolite of Clostridium perfringens type A, palmitic acid, enhances porcine enteric coronavirus porcine epidemic diarrhea virus infection

The host intestinal microbiota has emerged as the third element in the interactions between hosts and enteric viruses, and potentially affects the infection processes of enteric viruses. However, the interaction of porcine enteric coronavirus with intestinal microorganisms during infection remains unclear. In this study, we used 16S-rRNA-based Illumina NovaSeq high-throughput sequencing to identify the changes in the intestinal microbiota of piglets mediated by porcine epidemic diarrhea virus (PEDV) infection and the effects of the alterations in intestinal bacteria on PEDV infection and its molecular mechanisms. The intestinal microbiota of PEDV-infected piglets had significantly less diversity than the healthy group and different bacterial community characteristics. Among the altered intestinal bacteria, the relative abundance of Clostridium perfringens was significantly increased in the PEDV-infected group. A strain of C. perfringens type A, named DQ21, was successfully isolated from the intestines of healthy piglets. The metabolites of swine C. perfringens type A strain DQ21 significantly enhanced PEDV replication in porcine intestinal epithelial cell clone J2 (IPEC-J2) cells, and PEDV infection and pathogenicity in suckling piglets. Palmitic acid (PA) was identified as one of those metabolites with metabolomic technology, and significantly enhanced PEDV replication in IPEC-J2 cells and PEDV infection and pathogenicity in suckling piglets. PA also increased the neutralizing antibody titer in the immune sera of mice. Furthermore, PA mediated the palmitoylation of the PEDV S protein, which improved virion stability and membrane fusion, thereby enhancing viral infection. Overall, our study demonstrates a novel mechanism of PEDV infection, with implications for PEDV pathogenicity.

LPLUNC1 reduces glycolysis in nasopharyngeal carcinoma cells through the PHB1-p53/c-Myc axis.

Cancer cells prefer glycolysis to support their proliferation. Our previous studies have shown that the long palate, lung, and nasal epithelial cell clone 1 (LPLUNC1) can upregulate prohibitin 1 (PHB1) expression to inhibit the proliferation of nasopharyngeal carcinoma (NPC) cells. Given that PHB1 is an important regulator of cell energy metabolism, we explored whether and how LPLUNC1 regulated glucose glycolysis in NPC cells. LPLUNC1 or PHB1 overexpression decreased glycolysis and increased oxidative phosphorylation (OXPHOS)-related protein expression in NPC cells, promoting phosphorylated PHB1 nuclear translocation through 14-3-3σ. LPLUNC1 overexpression also increased p53 but decreased c-Myc expression in NPC cells, which were crucial for the decrease in glycolysis and increase in OXPHOS-related protein expression induced by LPLUNC1 overexpression. Finally, we found that treatment with all-trans retinoic acid (ATRA) reduced the viability and clonogenicity of NPC cells, decreased glycolysis, and increased OXPHOS-related protein expression by enhancing LPLUNC1 expression in NPC cells. Therefore, the LPLUNC1-PHB1-p53/c-Myc axis decreased glycolysis in NPC cells, and ATRA upregulated LPLUNC1 expression, ATRA maybe a promising drug for the treatment of NPC.

α and β catalytic subunits of cAMP‐dependent protein kinase regulate formoterol‐induced inflammatory gene expression changes in human bronchial epithelial cells

It has been proposed that genomic mechanisms contribute to adverse effects often experienced by asthmatic subjects who take regular, inhaled β2 -adrenoceptor agonists as a monotherapy. Moreover, data from preclinical models of asthma suggest that these gene expression changes are mediated by β-arrestin-2 rather than PKA. Herein, we tested this hypothesis by comparing the genomic effects of formoterol, a β2 -adrenoceptor agonist, with forskolin in human primary bronchial epithelial cells (HBEC). Gene expression changes were determined by RNA-sequencing. Gene silencing and genome editing were employed to explore the roles of β-arrestin-2 and PKA. The formoterol-regulated transcriptome in HBEC treated concurrently with TNFα was defined by 1480 unique gene expression changes. TNFα-induced transcripts modulated by formoterol were annotated with enriched gene ontology terms related to inflammation and proliferation, notably "GO:0070374~positive regulation of ERK1 and ERK2 cascade," which is an apparentβ-arrestin-2 target. However, expression of the formoterol- and forskolin-regulated transcriptomes were highly rank-order correlated and the effects of formoterol on TNFα-induced inflammatory genes were abolished by an inhibitor of PKA. Furthermore, formoterol-induced gene expression changes in BEAS-2B bronchial epithelial cell clones deficient in β-arrestin-2 were comparable with those expressed by their parental counterparts. Contrariwise, gene expression was partially inhibited in clones lacking the α-catalytic subunit (Cα) of PKA and abolished following the additional knockdown of the β-catalytic subunit (Cβ) paralogue. The effects of formoterol on inflammatory gene expression in airway epithelia are mediated by PKA and involve the cooperation of PKA-Cα and PKA-Cβ.

Molecular Cytogenetics Reveals Mosaicism in Human Umbilical Vein Endothelial Cells

Primary human umbilical vein endothelial cells (HUVECs) are consistently the most reliable in vitro model system for studying the inner lining of blood and lymphatic vessels or the endothelium. Primary human cells originate from freshly isolated tissues without genetic manipulation and generally show a modal number of 46 chromosomes with no structural alterations, at least during early passages. We investigated the cytogenetic integrity of HUVECs with conventional (G-banding) and molecular cytogenetic methods (spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH)). Our G-band data shows two X-chromosomes, confirming these HUVECs originate from a female donor. Notably, some cells consistently exhibit an unfamiliar banding pattern on one X chromosome toward the distal end of the long arm (Xq). Our FISH analysis confirms that approximately 50% of these HUVECs have a deletion of the Xq terminal region. SKY analysis indicates that the deleted region is apparently not integrated into any other chromosome. Finally, we demonstrated the presence of a similar Xq deletion in the daughter cell line, EA.hy926, which was generated by fusing HUVECs with A549 (a thioguanine-resistant clone of adenocarcinomic human alveolar basal epithelial cells). These findings will advance comprehension of HUVECs biology and will augment future endothelial studies.

Effects of Dexamethasone on DNA Synthesis in Lens Epithelial Cells Are Dependent on Cell Type and Growth Factor

Purpose To determine the factors that influence the ability of dexamethasone (dex) to inhibit or stimulate the growth of lens epithelial cells. Method Different growth factors with or without dex (10−6 M) were added to quiescent cultures of two clones of Nakano mouse lens epithelial cells (NK11) in serum-free medium. DNA synthesis was then measured after 8–12 hours by the incorporation of tritiated thymidine. Results Dex was found to both stimulate and inhibit mitogen-induced 3H-thymidine incorporation into the DNA of cultured mouse lens epithelial cells. Enhancement or repression by dex was found to depend on the growth factor used to stimulate the quiescent cell. EGF and insulin were consistently inhibited with dex. Basic fibroblast growth factor (bFGF) and retinoblastoma-derived growth factor (RbDGF) were both enhanced and inhibited by dex, depending on the growth factor concentration and the cell clone used for the experiment. Additionally, RbDGF protects against the dex inhibition of insulin stimulation, but not the inhibition of EGF stimulation. Progesterone, an inhibitor of the activation of the glucocorticoid receptor, blocks the dex inhibitory effect on the EGF and insulin stimulation of DNA synthesis. The ability of progesterone to affect the dex inhibition is consistent with the dex receptor modulating DNA synthesis. The dex effect on DNA synthesis, either stimulatory or inhibitory, was still seen if dex was added as late as 10 hours after the growth factor. Conclusions The study demonstrated that dex reduces the overall growth and activity of lens epithelial cells in vitro. This result provides insight into the risk of developing posterior subcapsular cataracts (PSC) in patients on oral glucocorticoid therapy. Understanding the basic mechanisms by which steroids mediate lens cell growth may provide the ability to more accurately predict who will develop PSC. The present studies show the difference in the effect of dex from lens cell to lens cell, but, more importantly, suggest a pattern of dependent variables that might prove useful in such predictions.

Ginsenoside Rk2 Protects against Ulcerative Colitis via Inactivating ERK/MEK Pathway by SIRT1.

Chinese traditional medicine is widely used in the treatment of ulcerative colitis (UC). Ginsenoside Rk2 is a newly discovered dammarane triterpenoid saponin isolated from ginseng. Our study aimed to investigate the effects of Ginsenoside Rk2 on UC. Human clones of colorectal adenocarcinoma Caco-2 cells and human intestinal epithelial THP-1 cells were co-cultured to establish a UC model in vitro. Cell viability and apoptosis were analyzed by cell counting kit 8 (CCK-8) and flow cytometry assay, respectively. Inflammatory cytokines' mRNA levels were measured by real-time quantitative polymerase chain reaction (RT-qPCR). Western blot was applied to examine the protein expression of apoptosis-associated proteins and the activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MEK) pathway. Furthermore, fisetin, an ERK kinase activator, was used to carry out rescue experiment. SRT1720, an activator of SIRT1, was applied to increase the SIRT1 protein levels while SIRT1 inhibitor nicotinamide (NAM) exerted the opposite effect. Ginsenoside Rk2 promoted cell viability, suppressed cell apoptosis, and reduced the release of pro-inflammatory cytokines including interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor-α (TNF-α) of HT-29 cells in UC model in a concentration-dependent manner. Meanwhile, the inhibitory effects of Ginsenoside Rk2 on the ERK/MEK pathway strengthened with the increase of concentration, and was verified by fisetin application. Furthermore, the upregulation of SIRT1 induced by Ginsenoside Rk2 prompted dephosphorylation of ERK and MEK to attenuate ERK/MEK pathway activation and reduced inflammatory progress, which was confirmed by SRT1720 as well as NAM. Ginsenoside Rk2 inactivated ERK/MEK pathway by regulating SIRT1 to restore the cellular function of human intestinal epithelial THP-1 cells. Therefore, Ginsenoside Rk2 may be effective in the treatment of UC.

Prostaglandin E2 Enhances Gap Junctional Intercellular Communication in Clonal Epithelial Cells.

Prostaglandins are a group of lipids that produce diverse physiological and pathological effects. Among them, prostaglandin E2 (PGE2) stands out for the wide variety of functions in which it participates. To date, there is little information about the influence of PGE2 on gap junctional intercellular communication (GJIC) in any type of tissue, including epithelia. In this work, we set out to determine whether PGE2 influences GJIC in epithelial cells (MDCK cells). To this end, we performed dye (Lucifer yellow) transfer assays to compare GJIC of MDCK cells treated with PGE2 and untreated cells. Our results indicated that (1) PGE2 induces a statistically significant increase in GJIC from 100 nM and from 15 min after its addition to the medium, (2) such effect does not require the synthesis of new mRNA or proteins subunits but rather trafficking of subunits already synthesized, and (3) such effect is mediated by the E2 receptor, which, in turn, triggers a signaling pathway that includes activation of adenylyl cyclase and protein kinase A (PKA). These results widen the knowledge regarding modulation of gap junctional intercellular communication by prostaglandins.

Isolation and Characterization of Mammalian Otic Progenitor Cells that Can Differentiate into Both Sensory Epithelial and Neuronal Cell Lineages.

The mammalian inner ear mediates hearing and balance and during development generates both cochleo-vestibular ganglion neurons and sensory epithelial receptor cells, that is, hair cells and support cells. Cell marking experiments have shown that both hair cells and support cells can originate from a common progenitor. Here, we demonstrate the lineage potential of individual otic epithelial cell clones using three cell lines established by a combination of limiting dilution and gene-marking techniques from an embryonic day 12 (E12) rat otocyst. Cell-type specific marker analyses of these clonal lines under proliferation and differentiation culture conditions demonstrate that during differentiation immature cell markers (Nanog and Nestin) were downregulated and hair cell (Myosin VIIa and Math1), support cell (p27Kip1 and cytokeratin) and neuronal cell (NF-H and NeuroD) markers were upregulated. Our results suggest that the otic epithelium of the E12 mammalian inner ear possess multipotent progenitor cells able to generate cell types of both sensory epithelial and neural cell lineages when cultured under a differentiation culture condition. Understanding the molecular mechanisms of proliferation and differentiation of multipotent otic progenitor cells may provide insights that could contribute to the development of a novel cell therapy with a potential to initiate or stimulate the sensorineural repair of damaged inner ear sensory receptors. Anat Rec, 303:451-460, 2020. © 2019 American Association for Anatomy.

Infection of Human Tracheal Epithelial Cells by H5 Avian Influenza Virus Is Regulated by the Acid Stability of Hemagglutinin and the pH of Target Cell Endosomes.

Despite the possible relationships between tracheal infection and concomitant infection of the terminal part of the lower respiratory tract (bronchioles/alveoli), the behavior of avian influenza viruses (AIVs), such as H5N1, in the conducting airways is unclear. To examine the tropism of AIVs for cells lining the conducting airways of humans, we established human tracheal epithelial cell clones (HTEpC-Ts) and examined their susceptibility to infection by AIVs. The HTEpC-Ts showed differing susceptibility to H5N1 and non-zoonotic AIVs. Viral receptors expressed by HTEpC-Ts bound all viruses; however, the endosomal pH was associated with the overall susceptibility to infection by AIVs. Moreover, H5N1 hemagglutinin broadened viral tropism to include HTEpC-Ts, because it had a higher pH threshold for viral–cell membrane fusion. Thus, H5N1 viruses infect human tracheal epithelial cells as a result of their higher pH threshold for membrane fusion which may be one mechanism underlying H5N1 pathogenesis in human airway epithelia. Efficient replication of H5N1 in the conducting airways of humans may facilitate infection of the lower respiratory tract.

Cyclin E Overexpression in Human Mammary Epithelial Cells Promotes Epithelial Cancer-Specific Copy Number Alterations.

SummaryCyclin E, a key cell cycle regulatory protein, has been linked to oncogenesis when dysregulated. We have previously shown that overexpression of cyclin E causes replication stress, leading to failure to complete replication at specific chromosomal loci during S phase of the cell cycle. This in turn promotes chromosomal damage during anaphase. Here we show that non-transformed human mammary epithelial cell clones that survive such aberrant mitoses have a specific and reproducible pattern of chromosomal Copy Number Alterations (CNAs) that we have characterized and termed the cyclin E CNA signature. Using a number of computational approaches, we show that this signature resembles one specific CNA pattern enriched in differentiated epithelial-like tumors of the breast and ovary. Analysis of the CNA profile of these clones provides a potential mechanism for cyclin E-mediated oncogenesis.

Expression of a neoantigen renders pancreas cancer transiently susceptible to immunotherapy

Abstract Immune checkpoint blockade has been largely ineffective for treating pancreatic ductal adenocarcinoma (PDA). Here, we develop a novel model of pancreatic cancer to investigate reasons for response and failure to immune checkpoint blockade in this lethal malignancy. Primary tumor epithelial cell clones from the genetically engineered KPC mouse model transduced to express a specific luciferase antigen, commonly used for bioluminescent in mice, rapidly establish tumors similar to parental cells after orthotopic implantation into syngeneic mice. Unexpectedly, αPD-L1 or αPD-1, which fail to elicit responses to luciferase- KPC parental cells, induce objective responses to luciferase+ KPC clones and prolong animal survival. We identify the immunodominant luciferase H- 2Db-restricted epitope and generate a fluorescently labeled peptide:MHC tetramer that specifically binds PDA-specific CD8 T cells. Up to 40% of tumor-infiltrating T cells bind this tetramer, yet most of these cells co-express PD-1 and Lag-3 and are dysfunctional. αPD-L1 enhances the systemic expansion of tetramer+ T cells and induced the accumulation of PD-1+ Ki67+ and Ki67- tetramer+ T cells in PDA. αPD-L1 significantly reduced the frequency of intra-tumoral tetramer+ T cells that expressed Lag-3 resulting in enhanced ex vivo peptide-specific cytokine production. However, tumors rapidly returned despite T cell infiltration and prolonged immunotherapy. Current efforts are underway to study the evolution of PDA-specific T cells and tumor clones using single cell technologies to inform clinical immunotherapy design.

Development and biochemical and immunological characterization of early passage and immortalized bovine intestinal epithelial cell lines from the ileum of a young calf.

The intestinal epithelium is a major site of interaction with pathogens. In bovine intestinal epithelial cells (BIECs), Toll-like receptors (TLRs) play an important role in innate immune responses against enteric pathogens. This study is aimed at establishing a stable bovine intestinal epithelial cell line that can be maintained by a continuous passage so that studies on innate immune responses against various enteric pathogens can be performed. The main goal was to establish pure cultures of primary and immortalized bovine intestinal epithelial cells from the ileum and then characterize them biochemically and immunologically. Mixed epithelial and fibroblast bovine ileal intestinal cultures were first established from a 2-day old calf. Limiting dilution method was used to obtain a clone of epithelial cells which was characterized using immunocytochemistry (ICC). The selected clone BIEC-c4 was cytokeratin positive and expressed low levels of vimentin, confirming the epithelial cell phenotype. Early passage BIEC-c4 cells were transfected with either simian virus 40 (SV40) large T antigen or human telomerase reverse transcriptase (hTERT), or human papillomavirus (HPV) type 16E6/E7 genes to establish three immortalized BIEC cell lines. The expression of SV40, hTERT and HPV E6/E7 genes in immortalized BIECs was confirmed by a polymerase chain reaction (PCR). Immunocytochemistry and immunofluorescence assays also confirmed the expression of SV40, hTERT and HPV E6 proteins. The immortalized BIECs were cytokeratin positive and all except HPV-BIECs expressed low levels of vimentin. A growth kinetics study indicated that there were no significant differences in the doubling time of immortalized BIECs as compared to early passage BIEC-c4 cells. All four BIEC types expressed TLR 1-10 genes, with TLR 3 and 4 showing higher expression across all cell types. These newly established early passage and immortalized BIEC cell lines should serve as a good model for studying infectivity, pathogenesis and innate immune responses against enteric pathogens.

Src Cooperates with Oncogenic Ras in Tumourigenesis via the JNK and PI3K Pathways in Drosophila epithelial Tissue.

The Ras oncogene (Rat Sarcoma oncogene, a small GTPase) is a key driver of human cancer, however alone it is insufficient to produce malignancy, due to the induction of cell cycle arrest or senescence. In a Drosophila melanogaster genetic screen for genes that cooperate with oncogenic Ras (bearing the RasV12 mutation, or RasACT), we identified the Drosophila Src (Sarcoma virus oncogene) family non-receptor tyrosine protein kinase genes, Src42A and Src64B, as promoting increased hyperplasia in a whole epithelial tissue context in the Drosophila eye. Moreover, overexpression of Src cooperated with RasACT in epithelial cell clones to drive neoplastic tumourigenesis. We found that Src overexpression alone activated the Jun N-terminal Kinase (JNK) signalling pathway to promote actin cytoskeletal and cell polarity defects and drive apoptosis, whereas, in cooperation with RasACT, JNK led to a loss of differentiation and an invasive phenotype. Src + RasACT cooperative tumourigenesis was dependent on JNK as well as Phosphoinositide 3-Kinase (PI3K) signalling, suggesting that targeting these pathways might provide novel therapeutic opportunities in cancers dependent on Src and Ras signalling.

Renal interstitial cells support proximal tubular regeneration in response to local injury

Platelet‐derived growth factor chain B (PDGF‐B) and PDGF receptor β (PDGFRβ)‐signalling contribute to epithelial‐interstitial cell communication in many organs and facilitates regenerative functions, such as wound healing and tissue repair. In the kidney, a potential role of interstitial cells in tubular regeneration remains obscure. Here we determined the role of interstitial PDGFRβ+ cells in tubular regeneration by serial intravital multiphoton microscopy and subsequent3‐D reconstruction. We used inducible PDGFRβ‐Cre x Confetti mice, to identify PDGFRβ+ interstitial cells in vivo by membrane‐targeted CFP, nuclear GFP and cytosolic YFP and RFP‐expression. PDGFRβ‐Cre x GCamP5 mice were used to determine intracellular calcium [Ca2+]of interstitial cells. Baseline imaging revealed interstitial PDGFRβ+ cells with dendritic morphology and long cell extensions connecting individual PDGFRβ+ cells and the tubular epithelium. Local tubular injury of 1–2 proximal tubule (PT) cells was induced by high laser exposure and caused an immediate 2‐fold increase of [Ca2+]in those PDGFRβ+ cells, which were located adjacent to the affected PT (n=40, p<.0001). The increase in interstitial PDGFRβ [Ca2+] was unaltered by suramin pre‐treatment (50 mg/kg), remained elevated over the first 72h post injury, and was associated with PDGFRβ‐cell proliferation (monochromatic cell division in PDGFRβ‐Crex Confetti mice). Furthermore, we observed the rapid migration (37.81±5 μm/24 h) of interstitial cells towards the injury site. Within 12–24 h, the injury site was enclosed by recruited PDGFRβ+interstitial cells. The recruitment of interstitial cells to the injury site was accompanied by the flattening of the resident tubular cells, followed by clonal epithelial cell division starting 3 days after injury. The latter was revealed by Pax8 x Confetti mice, which express CFP, GFP, YFP or RFP in all tubular cells. Conversely, we obtained no evidence for the integration of extra‐tubular cells into the tubular epithelium. To further investigate the tubular cell regeneration in response to interstitial cell recruitment, we performed histological analysis of the affected kidney areas. 24 h after the injury, the affected tubular epithelium expressed increased PDGF‐B levels. After 48 h, we found dedifferentiated tubular epithelial cells in the vicinity of the injury site (positive CD44‐staining). Finally, 7 days post injury, the recruited interstitial cells had moved a way from the injury site and had dispersed in the interstitium. There generated PT cells stained positive for CD44 and megalin, and reached 50% of control PT‐cell albumin uptake, as determined in vivo. In summary, our data suggest that renal interstitial cells are motile. Thus, local PT cell injury led to an immediate activation and recruitment of interstitial cells to the injury site. Proximal tubular regeneration was exclusively facilitated by clonal cell division of dedifferentiated resident epithelial cells, suggesting that interstitial cells modulate tubular regeneration rather than integrate into the epithelium. Together, these data suggest a communication between the interstitium and the tubular system, which appears to be functionally relevant for tubular regeneration.Support or Funding InformationDeutsche Forschungsgemeinschaft (SFB699/B7)

Establishment and Characterization of an SV40 Large T Antigen-Transduced Porcine Colonic Epithelial Cell Line

Continuous cell lines have become indispensable tools that have enabled investigations into cellular mechanisms by increasing experimental reproducibility and sample availability, and decreasing the use of experimental animals. To facilitate studies of epithelial barrier function of the porcine colon, we aimed to establish an epithelial cell line with an extended replicative capacity. Cells were isolated from the proximal colon of a 3-week-old piglet and transduced using a recombinant retroviral vector construct containing the simian virus 40 large T antigen (SV40 TAg). We established a clonal epithelial cell line, referred to as PoCo83-3, that stably expressed the SV40 TAg, verified at mRNA and protein levels. PoCo83-3 showed epithelial cell-specific features, such as cobblestone-like morphology, dome structure formation, the presence of apical microvilli, and the expression of keratin 18, E-cadherin and the tight junction-associated proteins zonula occludens-1, occludin, and claudin-1. To validate PoCo83-3 as an in vitro model in epithelial barrier research, proinflammatory cytokine-inducible alterations in barrier integrity were demonstrated by incubating the cells with TNF-α and IFN-γ for 48 h. These cytokine treatments promoted a decreased transepithelial electrical resistance. In summary, PoCo83-3 exhibited an extended life span and a differentiated phenotype while maintaining epithelial characteristics. Based on these results, we present this cell line as a valuable in vitro model for investigations of epithelial barrier function in the porcine colon.

Establishment of primary bovine intestinal epithelial cell culture and clone method.

The aim of this study was to establish bovine intestinal epithelial cell (BIEC) line and provide a novel clone cell method. Although various strategies of bovine cell culture and clone techniques have been reported, these methods remain not established. Here, we culture successfully primary BIECs and establish a novel clone cell method. Our result showed that BIECs could be successfully cultured and passaged about generation 5. These cellular aggregates and clusters were adherent loosely at day 2 of culture. Cell aggregates and clusters start to proliferate after approximately 4d. The BIECs showed positive reaction against cytokeratin 18, E-cadherin, and characteristics of epithelial-like morphology. In addition, the fatty acid-binding proteins (FABPs), villin, and intestinal peptidase (IP) band were positive in BIECs. Our results suggest that the establishment of culturing and clone BIEC methods will apply to isolate and clone other primary cells. These BIECs could therefore contribute to the study of bovine intestinal nutrient absorption and regulation, immune regulation, and the pathogenesis of the bovine intestinal disease, which will provide intestinal cell model in vitro.

The Force Dynamics of Interacting Cells

We have developed a method to understand the forces that pairs of cells exert upon each other on a surface. When exerting a force or torque on another object, a cell experiences an equal and opposite recoil force, and must therefore brace itself against its substrate. If the substrate is soft and elastic, then absorbing the recoil causes observable displacements of marker particles imbedded below its surface. We here describe a method of traction force microscopy, which can detect and analyze the recoil displacements. We validate our method using a clone of Breast Ductal Epithelial cells, (BDE's), that crawl and interact on collagen coated polyacrylamide substrates of different stiffness. For these cells, noise in the bead data causes random, or “phantom”, forces that fluctuate with standard deviation of approximately 10 nN. In cases where cells are touching, we universally observe significant tension forces that are usually aligned with the centroid-centroid axis of the cell pair. In contrast there are only a few isolated cases where shear forces or torques between BDE cells are above threshold. We also observe that the BDE's exert tension forces in two very different ways; first as part of a “grappling” process which brings the cell membranes into greater contact, and second as the basis for a “tug-of-war,” which has the opposite result. Grappling seems to happen only in the early stages as BDE's pull themselves into alignment. Later on, during the tug-of-war, the cell-cell tension grows and can reach peak values of over 200 nN. In addition, the cell-substrate adhesions near the contact zone are lost.

Decreased eIF3e Expression Can Mediate Epithelial-to-Mesenchymal Transition through Activation of the TGFβ Signaling Pathway.

The eIF3e protein is a component of the multisubunit eIF3 complex, which is essential for cap-dependent translation initiation. Decreased eIF3e expression is often observed in breast and lung cancer and has been shown to induce epithelial-to-mesenchymal transition (EMT) in breast epithelial cells by an unknown mechanism. Here, we study the effect of decreased eIF3e expression in lung epithelial cells by creating stable clones of lung epithelial cells (A549) that express an eIF3e-targeting shRNA. Our data indicate that decreased eIF3e expression in lung epithelial cells leads to EMT, as it does in breast epithelial cells. Importantly, we show that decreased eIF3e expression in both lung and breast epithelial cells leads to the overproduction of the TGFβ cytokine and that inhibition of TGFβ signaling can reverse eIF3e-regulated EMT in lung epithelial cells. In addition, we discovered that several mRNAs that encode important EMT regulators are translated by a cap-independent mechanism when eIF3e levels are reduced. These findings indicate that EMT mediated by a decrease in eIF3e expression may be a general phenomenon in epithelial cells and that it requires activation and maintenance of the TGFβ signaling pathway. These results indicate that inhibition of TGFβ signaling could be an efficient way to prevent metastasis in patients with NSCLC that display reduced eIF3e expression.