scholarly journals 2325

2017 ◽  
Vol 1 (S1) ◽  
pp. 61-62
Author(s):  
Evan Brady Lynch ◽  
Tatiana Goretsky ◽  
Emily Bradford ◽  
Tianyan Gao ◽  
Terrence Barrett
Keyword(s):  
Stem Cell ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Cultures ◽  
Steroid Therapy ◽  
Target Genes ◽  
Intestinal Epithelial Cells ◽  
Stem Cell Markers ◽  
Intestinal Epithelial ◽  
Ru 486

OBJECTIVES/SPECIFIC AIMS: Intestinal stem cells (ISC) primarily act in the repair of ulcerated epithelium, and their proliferative capacity relies on Wnt/β-catenin signaling. However, the role of GCs on basal epithelial cell signaling has not been fully characterized. The objective of this study was to interrogate a mechanism by which steroids may limit ISC activation. GCs inhibit NFκB signaling, which has been shown to play a role in nuclear β-catenin activation in epithelial cells. We hypothesized that GCs limit Wnt/β-catenin signaling required for ISC activation and epithelial restitution by inhibiting NFκB activation in epithelial cells. METHODS/STUDY POPULATION: To examine the effects of GCs on intestinal epithelial cells, we treated a nontransformed human colonic epithelial cell line (NCM460) with dexamethasone and observed the effects on NFκB and Wnt/β-catenin signaling events. We isolated mouse epithelial cells from the distal colon for stem cell culture as 3D “organoids.” We obtained pure epithelial cell preparations from mucosal biopsies isolated from patients treated at GI clinics at the University of Kentucky Chandler Hospital and VA Medical Center, Lexington. Steroid treated patients with equivalent levels of inflammation, but no mucosal ulceration were used as controls. RESULTS/ANTICIPATED RESULTS: In steroid-treated NCM460 cells, we saw an increase in steroid-responsive genes GILZ and SGK1. We saw a significant decrease in transcripts for Wnt target genes, including Axin2 and cmyc; NFκB target genes, including IFNG and IL6; and the shared NFκB and Wnt pathway co-activator CREBBP, despite unchanged transcript levels for β-catenin (CTNNB1). This data was corroborated in 3D stem cell cultures from cells isolated from mouse colon tissue, which had significant decreases in transcripts for stem cell markers Lgr5 and Ascl2, proliferative markers KI67 and PCNA, and Wnt target Axin2. NCM460s transfected with a lentivirus carrying a TCF/LEF luciferase construct showed a 2.5-fold decrease in TNF-stimulated luciferase activity with dexamethasone treatment. Interestingly, this effect can be rescued by glucocorticoid receptor (GR) blockade with RU-486. Intestinal epithelial cells from patient biopsies showed significant decreases in colitis-induced Axin2, p-LRP6 (a positive marker of Wnt Signaling) and nuclear β-catenin, which correlated with decreased p-p65 protein levels. DISCUSSION/SIGNIFICANCE OF IMPACT: Together, these data suggest that steroid therapy inhibits Wnt/β-catenin signaling at multiple levels, and effects stem cell proliferation in pure stem cell cultures. Decreases in TCF/LEF transcriptional activation (nuclear β-catenin’s DNA binding target) can be reversed with steroid receptor blockade with RU-486, suggesting that a receptor level interaction may be occurring. Interestingly, the required co-activator CBP, shared between NFκB and Wnt pathways, has decreased transcription following steroid treatment, which may provide a mechanism for limited Wnt activation following steroid therapy. Although steroids play a significant role in regulating the amount of inflammatory damage that occurs during IBD treatment, our data suggest that they may be limiting pathways required for effective healing as well.

scholarly journals Amelioration of Congenital Tufting Enteropathy in EpCAM (TROP1)-Deficient Mice via Heterotopic Expression of TROP2 in Intestinal Epithelial Cells

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1847
Author(s):  
Gaku Nakato ◽  
Sohshi Morimura ◽  
Michael Lu ◽  
Xu Feng ◽  
Chuanjin Wu ◽  
...  
Keyword(s):  
Stem Cell ◽  
Epithelial Cells ◽  
Cell Function ◽  
Intestinal Epithelial Cells ◽  
Surface Proteins ◽  
Stem Cell Markers ◽  
Intestinal Epithelial ◽  
Mesenteric Lymph Nodes ◽  
Function Expression ◽  
Deficient Mice

TROP1 (EpCAM) and TROP2 are homologous cell surface proteins that are widely expressed, and often co-expressed, in developing and adult epithelia. Various functions have been ascribed to EpCAM and TROP2, but responsible mechanisms are incompletely characterized and functional equivalence has not been examined. Adult intestinal epithelial cells (IEC) express high levels of EpCAM, while TROP2 is not expressed. EpCAM deficiency causes congenital tufting enteropathy (CTE) in humans and a corresponding lethal condition in mice. We expressed TROP2 and EpCAM in the IEC of EpCAM-deficient mice utilizing a villin promoter to assess EpCAM and TROP2 function. Expression of EpCAM or TROP2 in the IEC of EpCAM knockout mice prevented CTE. TROP2 rescue (T2R) mice were smaller than controls, while EpCAM rescue (EpR) mice were not. Abnormalities were observed in the diameters and histology of T2R small intestine, and Paneth and stem cell markers were decreased. T2R mice also exhibited enlarged mesenteric lymph nodes, enhanced permeability to 4 kDa FITC-dextran and increased sensitivity to detergent-induced colitis, consistent with compromised barrier function. Studies of IEC organoids and spheroids revealed that stem cell function was also compromised in T2R mice. We conclude that EpCAM and TROP2 exhibit functional redundancy, but they are not equivalent.

scholarly journals Intestinal epithelial CD98 synthesis specifically modulates expression of colonic microRNAs during colitis

2012 ◽  
Vol 302 (11) ◽  
pp. G1282-G1291 ◽  
Author(s):  
Moiz A. Charania ◽  
Saravanan Ayyadurai ◽  
Sarah A. Ingersoll ◽  
Bo Xiao ◽  
Emilie Viennois ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Mirna Expression ◽  
Target Genes ◽  
Immune Cell ◽  
Intestinal Inflammation ◽  
Intestinal Epithelial Cells ◽  
Cell Types ◽  
Intestinal Epithelial ◽  
Small Noncoding Rnas

The transmembrane glycoprotein CD98 is known to be involved in intestinal inflammation. In the present study, we found that CD98 overexpression in intestinal epithelial cells does not normally affect the expression of colonic (epithelial and immune cell) microRNAs (miRNAs), small noncoding RNAs that posttranscriptionally regulate a wide variety of biological processes. However, upon dextran sulfate sodium (DSS) treatment, the expression of several colonic miRNAs, but not miRNAs from other tissues such as liver and spleen, were differentially regulated in mice overexpressing CD98 in epithelial cells compared with wild-type (WT) animals. For example, the level of colonic miRNA 132 was not affected by DSS treatment in WT animals but was upregulated in mice overexpressing CD98 in intestinal epithelial cells. Other colonic miRNAs, including colonic miRNA 23a and 23b, were downregulated in WT animals after DSS treatment but not in colonic epithelial cell CD98-overexpressing mice. Interestingly, the expression of potential miRNA target genes affected intestinal epithelial cells that overexpress CD98 and cell types that did not overexpress CD98 but were in close proximity to CD98-overexpressing intestinal epithelial cells. Taken together, these observations show that the combination of an inflammatory context and intestinal epithelial cell expression of CD98 affects the regulation of miRNA expression in colonic epithelial and immune cells. This is new evidence that protein expression modulates miRNA expression and suggests the existence of regulatory crosstalk between proteins and miRNAs in diseases such as colitis.

Studying Permeability in a Commonly Used Epithelial Cell Line: T84 Intestinal Epithelial Cells

2011 ◽  
pp. 115-137 ◽  
Author(s):  
Rino P. Donato ◽  
Adaweyah El-Merhibi ◽  
Batjargal Gundsambuu ◽  
Kai Yan Mak ◽  
Emma R. Formosa ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Line ◽  
Intestinal Epithelial Cells ◽  
Epithelial Cell Line ◽  
Intestinal Epithelial

Induction of endothelin-1 synthesis by IL-2 and its modulation of rat intestinal epithelial cell growth

1998 ◽  
Vol 275 (3) ◽  
pp. G556-G563 ◽  
Author(s):  
Takeharu Shigematsu ◽  
Soichiro Miura ◽  
Masahiko Hirokawa ◽  
Ryota Hokari ◽  
Hajime Higuchi ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Proliferative Response ◽  
Intestinal Epithelial Cell ◽  
Culture Media ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial

Endothelin (ET), a vasoconstrictive peptide, is known to have a variety of biological actions. Although ET is released by vascular endothelial cells, other cell populations also have been reported to synthesize and release ET. In this study, we examined whether ET is synthesized by intestinal epithelial cells and whether it affects induction of epithelial cell proliferation by interleukin-2 (IL-2). Subconfluent monolayers of intestinal epithelial cells (IEC-6 and IEC-18) were maintained in serum-free medium before addition of rat IL-2. Both IEC-6 and IEC-18 cells released ET-1 into the medium under unstimulated conditions, as determined by a sandwich ELISA. IL-2 significantly enhanced ET-1 release in a time-dependent manner. ET-3 was not detectable in the culture media of either cell line. Expression of ET-1 and ET-3 mRNA in epithelial cells was assessed by competitive PCR. Both cell lines were shown to express ET-1 mRNA, but no ET-3 mRNA was detected. IL-2 treatment enhanced ET-1 mRNA expression by both IEC-6 and IEC-18 cells. Both cell lines also expressed mRNA for ETA and ETB receptor subtypes. When cell proliferation was assessed, exogenous ET-1 induced a slight proliferative response in both types of cells that was consistent and significant at low ET-1 concentrations; cell growth was inhibited at a higher concentration (10−7M). IL-2 produced a significant proliferative response in both cell lines. However, the addition of ET-1 (10−7 M) to culture media attenuated the IL-2-induced increase in cell proliferation. ETA-receptor antagonists significantly enhanced cellular proliferation, suggesting involvement of the ETA receptor in modulation of IL-2-induced intestinal epithelial cell growth.

scholarly journals Human intestinal epithelial cell-induced CD8+ T cell activation is mediated through CD8 and the activation of CD8-associated p56lck.

1995 ◽  
Vol 182 (4) ◽  
pp. 1079-1088 ◽  
Author(s):  
Y Li ◽  
X Y Yio ◽  
L Mayer
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Intestinal Epithelial Cells ◽  
T Cell Proliferation ◽  
Intestinal Epithelial

The activation of CD8+ suppressor T cells by normal intestinal epithelial cells in antigen-specific or allogeneic mixed cell culture systems has significant implications for the regulation of mucosal immune responses. In this study, we found that the capacity of epithelial cells to induce CD8+ suppressor T cell activation appeared to be linked to the binding of CD8 molecules on the T cell surface. This appears to be mediated by a non-class I molecule expressed on the epithelial cell surface, which binds to CD8 and results in the activation of the CD8-associated src-like tyrosine kinase, p56lck. Epithelial cell-stimulated p56lck activation is an early event (in contrast to monocytes) and is essential for T cell activation, since proliferation could be completely abrogated by pretreatment of T cells with genestein or herbamycin, both of which are protein tyrosine kinase inhibitors. Pretreatment of T cells with anti-CD8 or of intestinal epithelial cells with an anti-epithelial cell mAb B9 inhibited p56lck activation and further confirmed that CD8 on the T cell and a CD8 ligand on the epithelial cell were involved in this T cell activation event. The specificity of this reaction was confirmed in experiments in which murine transfectants 3G4 and 3G8, expressing CD4 or CD8, respectively, were used. Coculture of 3G8 with epithelial cells but not with monocytes activated p56lck in this cell line, whereas p56lck was preferentially activated in 3G4 cells when monocytes were used as the stimulator cells. Although stimulation through CD8- and CD8-associated p56lck was important for epithelial cell-induced T cell activation, T cell proliferation could not be induced by cross-linking CD8 alone with monoclonal antibody anti-CD8. These data suggest that a second signal, possibly through the T cell antigen receptor since activation of the T cell receptor-associated kinase fyn was also seen, is required for epithelial cell-driven T cell proliferation.

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