scholarly journals SOX9 directly regulates IGFBP-4 in the intestinal epithelium

2013 ◽  
Vol 305 (1) ◽  
pp. G74-G83 ◽  
Author(s):  
Zhongcheng Shi ◽  
Chi-I Chiang ◽  
Toni-Ann Mistretta ◽  
Angela Major ◽  
Yuko Mori-Akiyama
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Epithelial Cells ◽  
Target Genes ◽  
Intestinal Epithelial Cells ◽  
Neutralizing Antibody ◽  
Intestinal Epithelial ◽  
Loss Of Function ◽  
Deficient Mice

SOX9 regulates cell lineage specification by directly regulating target genes in a discrete number of tissues, and previous reports have shown cell proliferative and suppressive roles for SOX9. Although SOX9 is expressed in colorectal cancer, only a few direct targets have been identified in intestinal epithelial cells. We previously demonstrated increased proliferation in Sox9-deficient crypts through loss-of-function studies, indicating that SOX9 suppresses cell proliferation. In this study, crypt epithelial cells isolated from Sox9-deficient mice were used to identify potential target genes of SOX9. Insulin-like growth factor (IGF)-binding protein 4 (IGFBP-4), an inhibitor of the IGF/IGF receptor pathway, was significantly downregulated in Sox9-deficient intestinal epithelial cells and adenoma cells of Sox9-deficient Apc Min/+ mice. Immunolocalization experiments revealed that IGFBP-4 colocalized with SOX9 in mouse and human intestinal epithelial cells and in specimens from patients with primary colorectal cancer. Reporter assays and chromatin immunoprecipitation demonstrated direct binding of SOX9 to the IGFBP-4 promoter. Overexpression of SOX9 attenuated cell proliferation, which was restored following treatment with a neutralizing antibody against IGFBP-4. These results suggest that SOX9 regulates cell proliferation, at least in part via IGFBP-4. Furthermore, the antiproliferative effect of SOX9 was confirmed in vivo using Sox9-deficient mice, which showed increased tumor burden when bred with Apc Min/+ mice. Our results demonstrate, for the first time, that SOX9 is a transcriptional regulator of IGFBP-4 and that SOX9-induced activation of IGFBP-4 may be one of the mechanisms by which SOX9 suppresses cell proliferation and progression of colon cancer.

Requirement of Notch activation during regeneration of the intestinal epithelia

2009 ◽  
Vol 296 (1) ◽  
pp. G23-G35 ◽  
Author(s):  
Ryuichi Okamoto ◽  
Kiichiro Tsuchiya ◽  
Yasuhiro Nemoto ◽  
Junko Akiyama ◽  
Tetsuya Nakamura ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Differentiation ◽  
Epithelial Cells ◽  
Notch Signaling ◽  
Tissue Regeneration ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Intestinal Epithelia ◽  
Notch Activation

Notch signaling regulates cell differentiation and proliferation, contributing to the maintenance of diverse tissues including the intestinal epithelia. However, its role in tissue regeneration is less understood. Here, we show that Notch signaling is activated in a greater number of intestinal epithelial cells in the inflamed mucosa of colitis. Inhibition of Notch activation in vivo using a γ-secretase inhibitor resulted in a severe exacerbation of the colitis attributable to the loss of the regenerative response within the epithelial layer. Activation of Notch supported epithelial regeneration by suppressing goblet cell differentiation, but it also promoted cell proliferation, as shown in in vivo and in vitro studies. By utilizing tetracycline-dependent gene expression and microarray analysis, we identified a novel group of genes that are regulated downstream of Notch1 within intestinal epithelial cells, including PLA2G2A, an antimicrobial peptide secreted by Paneth cells. Finally, we show that these functions of activated Notch1 are present in the mucosa of ulcerative colitis, mediating cell proliferation, goblet cell depletion, and ectopic expression of PLA2G2A, thereby contributing to the regeneration of the damaged epithelia. This study showed the critical involvement of Notch signaling during intestinal tissue regeneration, regulating differentiation, proliferation, and antimicrobial response of the epithelial cells. Thus Notch signaling is a key intracellular molecular pathway for the proper reconstruction of the intestinal epithelia.

scholarly journals Expression of lysophosphatidic acid receptor 5 is necessary for the regulation of intestinal Na+/H+ exchanger 3 by lysophosphatidic acid in vivo

2018 ◽  
Vol 315 (4) ◽  
pp. G433-G442 ◽  
Author(s):  
Kayte A. Jenkin ◽  
Peijian He ◽  
C. Chris Yun
Keyword(s):  
Epithelial Cells ◽  
Lysophosphatidic Acid ◽  
Direct Evidence ◽  
Intestinal Epithelial Cells ◽  
Regulatory Role ◽  
Intestinal Epithelial ◽  
Fluid Absorption ◽  
Wild Type

Lysophosphatidic acid (LPA) is a bioactive lipid molecule, which regulates a broad range of pathophysiological processes. Recent studies have demonstrated that LPA modulates electrolyte flux in the intestine, and its potential as an antidiarrheal agent has been suggested. Of six LPA receptors, LPA5 is highly expressed in the intestine. Recent studies by our group have demonstrated activation of Na+/H+ exchanger 3 (NHE3) by LPA5. However, much of what has been elucidated was achieved using colonic cell lines that were transfected to express LPA5. In the current study, we engineered a mouse that lacks LPA5 in intestinal epithelial cells, Lpar5ΔIEC, and investigated the role of LPA5 in NHE3 regulation and fluid absorption in vivo. The intestine of Lpar5ΔIEC mice appeared morphologically normal, and the stool frequency and fecal water content were unchanged compared with wild-type mice. Basal rates of NHE3 activity and fluid absorption and total NHE3 expression were not changed in Lpar5ΔIEC mice. However, LPA did not activate NHE3 activity or fluid absorption in Lpar5ΔIEC mice, providing direct evidence for the regulatory role of LPA5. NHE3 activation involves trafficking of NHE3 from the terminal web to microvilli, and this mobilization of NHE3 by LPA was abolished in Lpar5ΔIEC mice. Dysregulation of NHE3 was specific to LPA, and insulin and cholera toxin were able to stimulate and inhibit NHE3, respectively, in both wild-type and Lpar5ΔIEC mice. The current study for the first time demonstrates the necessity of LPA5 in LPA-mediated stimulation of NHE3 in vivo. NEW & NOTEWORTHY This study is the first to assess the role of LPA5 in NHE3 regulation and fluid absorption in vivo using a mouse that lacks LPA5 in intestinal epithelial cells, Lpar5ΔIEC. Basal rates of NHE3 activity and fluid absorption, and total NHE3 expression were not changed in Lpar5ΔIEC mice. However, LPA did not activate NHE3 activity or fluid absorption in Lpar5ΔIEC mice, providing direct evidence for the regulatory role of LPA5.

Progastrin1–80stimulates growth of intestinal epithelial cells in vitro via high-affinity binding sites

2003 ◽  
Vol 284 (2) ◽  
pp. G328-G339 ◽  
Author(s):  
P. Singh ◽  
X. Lu ◽  
S. Cobb ◽  
B. T. Miller ◽  
N. Tarasova ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Binding Sites ◽  
Intestinal Epithelial Cells ◽  
Full Length ◽  
Intestinal Epithelial ◽  
Growth Effects ◽  
High Affinity ◽  
Significant Difference

Proliferation and carcinogenesis of the large intestinal epithelial cells (IEC) cells is significantly increased in transgenic mice that overexpress the precursor progastrin (PG) peptide. It is not known if the in vivo growth effects of PG on IEC cells are mediated directly or indirectly. Full-length recombinant human PG (rhPG1–80) was generated to examine possible direct effects of PG on IEC cells. Surprisingly, rhPG (0.1–1.0 nM) was more effective than the completely processed gastrin 17 (G17) peptide as a growth factor. Even though IEC cells did not express CCK1and CCK2receptors (-R), fluorescently labeled G17 and Gly-extended G17 (G-Gly) were specifically bound to the cells, suggesting the presence of binding proteins other than CCK1-R and CCK2-R on IEC cells. High-affinity ( Kd= 0.5–1.0 nM) binding sites for125I-rhPG were discovered on IEC cells that demonstrated relative binding affinity for gastrin-like peptides in the order PG ≥ COOH-terminally extended G17 ≥ G-Gly > G17 > *CCK-8 (* significant difference; P< 0.05). In conclusion, our studies demonstrate for the first time direct growth effects of the full-length precursor peptide on IEC cells in vitro that are apparently mediated by the high-affinity PG binding sites that were discovered on these cells.

Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids

2021 ◽  
Author(s):  
Yu Takahashi ◽  
Yu Inoue ◽  
Keitaro Kuze ◽  
Shintaro Sato ◽  
Makoto Shimizu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Gene Expression Regulation ◽  
Intestinal Epithelial Cells ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Intestinal Epithelial

Abstract Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

7 LACTOBACILLUS REUTERI SUPPRESSES PRO-INFLAMMATORY DRIVEN REACTIVE OXYGEN SPECIES IN VITRO IN HUMAN INTESTINAL EPITHELIAL CELLS AND IN VIVO IN A TNBS COLITIS MOUSE MODEL

2020 ◽  
Vol 26 (Supplement_1) ◽  
pp. S41-S41 ◽  
Author(s):  
Wenly Ruan ◽  
Melinda Engevik ◽  
Alexandra Chang-Graham ◽  
Joseph Hyser ◽  
James Versalovic
Keyword(s):  
Reactive Oxygen Species ◽  
Epithelial Cells ◽  
Lactobacillus Reuteri ◽  
Intestinal Inflammation ◽  
Intestinal Epithelial Cells ◽  
Oxygen Species ◽  
Intestinal Epithelial ◽  
Colitis Model

Abstract Background Reactive oxygen species (ROS) play a role in maintaining intestinal epithelial homeostasis and are normally kept at low levels via antioxidant compounds. Dysregulation of ROS can lead to intestinal inflammation and contribute to inflammatory bowel disease (IBD). Select gut microbes possess the enzymatic machinery to produce antioxidants whereas others can dysregulate levels of ROS. Our model microbe, Lactobacillus reuteri (ATCC PTA 6475), has been demonstrated to reduce intestinal inflammation in mice models. It contains the genes encoding two distinct GshA-like glutamylcysteine ligases. We hypothesize that L. reuteri can secrete γ-glutamylcysteine to suppress ROS, minimize NFκB activation and regulate secretion of e pithelial cytokines. Methods & Results Conditioned media from L. reuteri was analyzed via mass spectrometry to confirm the presence of γ-glutamylcysteine. All cysteine containing products including γ-glutamylcysteine were fluorescently tagged in the conditioned media and then incubated with HT29 cell monolayers as well as human jejunal enteroid (HJE) monolayers. γ-glutamylcysteine was demonstrated to enter intestinal epithelial cells based on microscopy. Next, a Thioltracker assay was used to show increased intracellular glutathione levels by L. reuteri secreted γ-glutamylcysteine. HT29 cells and HJEs were then treated with IL-1β or hydrogen peroxide, and L. reuteri metabolites as well as γ-glutamylcysteine significantly suppressed pro-inflammatory cytokine driven ROS and IL-8 production. L. reuteri secreted products also reduced activity of NFκB as determined by a luciferase reporter assay. γ-glutamylcysteine deficient mutants were generated by targeted mutagenesis of GshA genes, and these mutant L. reuteri strains had a diminished ability to suppress IL-8 production and ROS. To further test the role of L. reuteri secreted γ-glutamylcysteine in vivo, a 2,4,6-Trinitrobenzenesulfonic acid (TNBS)- induced mouse colitis model was used. Adolescent mice were orogavaged with PBS, L. reuteri, L. reuteri GshA2 mutant, or γ-glutamylcysteine for a week after which TNBS was rectally administered to induce colitis. We demonstrate that L. reuteri and γ-glutamylcysteine can suppress histologic inflammation compared to PBS control and L. reuteri GshA2 mutant groups. Conclusions Together these data indicate that L. reuteri secretes γ-glutamylcysteine which can enter the intestinal epithelial cells and modulate epithelial cytokine production. It acts via suppression of ROS and NFκB which then decreases IL-8 production. We are able to demonstrate this in vitro in both HT 29 cells and HJEs. We now also demonstrate this in vivo in a mouse colitis model. These experiments highlight a prominent role for ROS intermediates in microbiome-mammalian cell signaling processes involved in immune responses and intestinal inflammation.

scholarly journals Decreased SLC26A3 expression and function in intestinal epithelial cells in response to Cryptosporidium parvum infection

2019 ◽  
Vol 317 (6) ◽  
pp. C1205-C1212 ◽  
Author(s):  
Anoop Kumar ◽  
Dulari Jayawardena ◽  
Arivarasu N. Anbazhagan ◽  
Ishita Chatterjee ◽  
Shubha Priyamvada ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cryptosporidium Parvum ◽  
Diarrheal Disease ◽  
Intestinal Epithelial Cells ◽  
Ex Vivo ◽  
Jejunal Mucosa ◽  
Intestinal Epithelial ◽  
Chloride Absorption ◽  
And Function

The protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a diarrheal disease worldwide. Infection in immunocompetent hosts typically results in acute, self-limiting, or recurrent diarrhea. However, in immunocompromised individuals infection can cause fulminant diarrhea, extraintestinal manifestations, and death. To date, the mechanisms underlying CP-induced diarrheal pathogenesis are poorly understood. Diarrheal diseases most commonly involve increased secretion and/or decreased absorption of fluid and electrolytes. We and others have previously shown impaired chloride absorption in infectious diarrhea due to dysregulation of SLC26A3 [downregulated in adenoma (DRA)], the human intestinal apical membrane Cl−/[Formula: see text] exchanger protein. However, there are no studies on the effects of CP infection on DRA activity. Therefore, we examined the expression and function of DRA in intestinal epithelial cells in response to CP infection in vitro and in vivo. CP infection (0.5 × 106 oocysts/well in 24-well plates, 24 h) of Caco-2 cell monolayers significantly decreased Cl−/[Formula: see text] exchange activity (measured as DIDS-sensitive 125I uptake) as well as DRA mRNA and protein levels. Substantial downregulation of DRA mRNA and protein was also observed following CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mucosa of C57BL/6 mice for 24 h. However, at 48 h after infection in vivo, the effects on DRA mRNA and protein were attenuated and at 5 days after infection DRA returned to normal levels. Our results suggest that impaired chloride absorption due to downregulation of DRA could be one of the contributing factors to CP-induced acute, self-limiting diarrhea in immunocompetent hosts.

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 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.

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