Hepatocyte nuclear factor-4α regulates expression of the serotonin transporter in intestinal epithelial cells

The serotonin transporter (SERT) functions to regulate the availability of serotonin (5-HT) in the brain and intestine. An intestine-specific mRNA variant arising from a unique transcription start site and alternative promoter in the SERT gene has been identified (iSERT; spanning exon 1C). A decrease in SERT is implicated in several gut disorders, including inflammatory bowel diseases (IBD). However, little is known about mechanisms regulating the iSERT variant, and a clearer understanding is warranted for targeting SERT for the treatment of gut disorders. The current studies examined the expression of iSERT across different human intestinal regions and investigated its regulation by HNF4α (hepatic nuclear factor-4α), a transcription factor important for diverse cellular functions. iSERT mRNA abundance was highest in the human ileum and Caco-2 cell line. iSERT mRNA expression was downregulated by loss of HNF4α (but not HNF1α, HNF1β, or FOXA1) in Caco-2 cells. Overexpression of HNF4α increased iSERT mRNA concomitant with an increase in SERT protein. Progressive promoter deletion and site-directed mutagenesis revealed that the HNF4α response element spans nucleotides −1,163 to −1150 relative to the translation start site. SERT mRNA levels in the intestine were drastically reduced in the intestine-specific HNF4α-knockout mice relative to HNF4αFL/FL mice. Both HNF4α and SERT mRNA levels were also downregulated in mouse model of ileitis (SAMP) compared with AKR control mice. These results establish the transcriptional regulation of iSERT at the gut-specific internal promoter (hSERTp2) and have identified HNF4α as a critical modulator of basal SERT expression in the intestine.

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Hepatocyte nuclear factor 4α regulates the expression of intestinal epithelial Na+/H+ exchanger isoform 3

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00225.2017 ◽

2018 ◽

Vol 314 (1) ◽

pp. G14-G21 ◽

Cited By ~ 3

Author(s):

Saminathan Muthusamy ◽

Jong Jin Jeong ◽

Ming Cheng ◽

Jessica A. Bonzo ◽

Anoop Kumar ◽

...

Keyword(s):

Promoter Activity ◽

Core Promoter ◽

Epithelial Cell Line ◽

Mrna Levels ◽

Nuclear Factor ◽

Intestinal Epithelial ◽

Hepatocyte Nuclear Factor ◽

Mobility Shift ◽

Protein Levels ◽

Hepatocyte Nuclear Factor 4Α

Na+/H+ exchanger isoform 3 (NHE3) plays a key role in coupled electroneutral NaCl absorption in the mammalian intestine. Reduced NHE3 expression or function has been implicated in the pathogenesis of diarrhea associated with inflammatory bowel disease (IBD) or enteric infections. Our previous studies revealed transcriptional regulation of NHE3 by various agents such as TNF-α, IFN-γ, and butyrate involving transcription factors Sp1 and Sp3. In silico analysis revealed that the NHE3 core promoter also contains a hepatocyte nuclear factor 4α (HNF-4α) binding site that is evolutionarily conserved in several species suggesting that HNF-4α has a role in NHE3 regulation. Nhe3 mRNA levels were reduced in intestine-specific Hnf4α-null mice. However, detailed mechanisms of NHE3 regulation by HNF-4α are not known. We investigated the regulation of NHE3 gene expression by HNF-4α in vitro in the human intestinal epithelial cell line C2BBe1 and in vivo in intestine-specific Hnf4α-null ( Hnf4αΔIEpC) and control ( Hnf4αfl/fl) mice. HNF-4α knockdown by short interfering RNA in C2BBe1 cells significantly decreased NHE3 mRNA and NHE3 protein levels. Gel mobility shift and chromatin immunoprecipitation assays revealed that HNF-4α directly interacts with the HNF-4α motif in the NHE3 core promoter. Site-specific mutagenesis on the HNF-4α motif decreased, whereas ectopic overexpression of HNF-4α increased, NHE3 promoter activity. Furthermore, loss of HNF-4α in Hnf4αΔIEpC mice decreased colonic Nhe3 mRNA and NHE3 protein levels. Our results demonstrate a novel role for HNF-4α in basal regulation of NHE3 expression. These studies represent an important and novel target for therapeutic intervention in IBD-associated diarrhea. NEW & NOTEWORTHY Our studies for the first time show that hepatocyte nuclear factor 4α directly regulates NHE3 promoter activity and its basal expression in the intestine.

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Elucidating the Importance and Regulation of Key Enhancers for Human MEIS1 Expression

Blood ◽

10.1182/blood-2021-151467 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3302-3302

Author(s):

Xining Yang ◽

Ping Xiang ◽

Leo Escano ◽

Ishpreet Dhillon ◽

Edith Schneider ◽

...

Keyword(s):

Overall Survival ◽

Cell Line ◽

Genome Editing ◽

Vector System ◽

Mrna Levels ◽

Start Site ◽

Translation Start Site ◽

Mass Spectrometry Experiment ◽

Enhancer Region ◽

Translation Start

Abstract Myeloid ecotropic virus insertion site 1 (MEIS1) is essential for normal hematopoiesis and is deregulated in a large subset of acute myeloid leukemia (AML) by yet unknown mechanisms. We previously identified 3 candidate enhancer regions: enhancer region 1 (E1) at -2 kb upstream; enhancer region 2 (E2) at +10.6 kb downstream inside intron 6; and enhancer region 3 (E3) +140 kb downstream of the translation start site. In the current study, we utilized CRISPR-Cas9 genome editing to further characterize these enhancers in a human AML cell line and identify the key transcription factors (TFs) associated with their function. To efficiently track MEIS1 expression levels, a GFP reporter, a P2A self-cleaving peptide tag and a hemagglutinin tag at its translation start site was introduced in a MEIS1 high expressing human AML cell line, U937. Then we introduced random mutations (Indels) along the MEIS1 locus utilizing a CRISPR-Cas9 mediated genome editing vector system in mono-allelic MEIS1-GFP-tagged U937 cells with special focus on the previously identified enhancer regions to find the key sequences important to the function of the MEIS1 enhancer regions. Two targeted regions yielding the highest proportion of GFP - cells corresponded to the E2 enhancer region within intron 6 and were referred to as E2.1 and E2.2. Using chromosome conformation capture (3C) assay, we detected a significantly decreased interaction (p=0.0022) between the promoter and the intron 6 region surrounding the E2 region in E2.2 targeted cells compared to the parental cells. Moreover, our data indicated that the DNA sequence within E2.2 is highly critical to this region's enhancer function which is further influenced by the larger genomic region surrounding the E2.1 gRNA targeted site. To identify TFs binding to the E2 region, we further scrutinized the E2.2 indel region for loss of TF binding sites. We performed TF prediction analysis and performed a protein pull down-mass spectrometry experiment to identify TF candidates. The overlap yielded a list of 7 TFs, each of which we targeted via CRISPR/Cas9. Reduction in GFP levels was only observed for FLI1 locus targeting but not for the other 6 TFs. Concordant reduction in MEIS1 and FLI1 levels were confirmed by immunoblotting. Additionally, chromatin immunoprecipitation (ChIP) followed by quantitative PCR revealed significant FLI1 enrichment at the promoter and at 3 sites surrounding the E2.2 region (p=0.0004) compared to 4 control regions scattered along the MEIS1 locus. Given a previous study indicating MEIS1 upregulation of FLI1 in normal hematopoiesis, we hypothesised that a positive feedback loop may exist between FLI1 and MEIS1 in AML. Since MEIS1 levels are frequently elevated in normal karyotype AML (CN-AML), we used the murine Hoxa9/Meis1 AML model as a surrogate for CN-AML and performed Meis1 ChIP-seq analysis. We detected direct Meis1 binding to the intronic region of the mouse Fli1 gene as well as other ETS factor loci, in Hoxa9/Meis1 cells. To better understand the clinical relevance of FLI1 in AML, we analyzed the Beat AML dataset. High FLI1 transcript levels correlated with adverse overall survival in CN-AML (p=0.044). Additionally, we observed a trend towards worse outcome with high FLI1 in the NPM1-mutated CN-AML subtype (p=0.069). We also observed a similar correlation in CN-AML for another ETS factor, ELF1, which we had previously shown to bind and upregulate MEIS1 expression in AML, suggesting a broader unrecognized role for ETS factors in AML. In summary, we have developed a rapid flow cytometry-based readout for the fine dissection and characterization of the cis-regulatory elements and associated TFs critical for MEIS1 transcription via CRISPR-Cas9 genetic manipulation. Our study revealed FLI1 as the candidate key regulator of MEIS1 expression and a positive correlation between FLI1 mRNA levels and worse overall survival in MEIS1-high AML subgroups. Disclosures No relevant conflicts of interest to declare.

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Regulation of nuclear factor-κB in intestinal epithelial cells in a cell model of inflammation

Mediators of Inflammation ◽

10.1080/09629350310001619681 ◽

2003 ◽

Vol 12 (5) ◽

pp. 277-283 ◽

Cited By ~ 16

Author(s):

Fadia R. Homaidan ◽

Iman Chakroun ◽

Marwan E. El-sabban

Keyword(s):

Epithelial Cells ◽

Intestinal Epithelial Cells ◽

Cell Model ◽

Interleukin 1 ◽

Nuclear Factor Κb ◽

Mrna Levels ◽

Nuclear Factor ◽

Intestinal Epithelial ◽

Mobility Shift ◽

Apoptotic Proteins

Background:Interleukin-1 (IL-1), an inflammatory cytokine whose levels are elevated in inflamed mucosa, causes part of its effect on intestinal epithelial cells (IEC) through inducing ceramide production.Aim:To study the role of nuclear factor-κB (NF-κB), a pro-inflammatory and anti-apoptotic factor, in IL-1- treated IEC.Methods:NF-κB activity and levels of apoptotic proteins were assessed by electrophoretic mobility shift assay and RNA-protection assay, respectively.Results:IL-1 and ceramide, which have been shown to partially mediate IL-l effects on IEC, activated NF-κB levels significantly. This activation was due to a decrease in IκB-α and IκB-β protein levels. Moreover, the ratio of mRNA levels of anti-apoptotic to pro-apoptotic proteins was significantly increased in IL-1-treated IEC.Conclusion:NF-κB may play a key role in the regulation of the expression of pro-inflammatory and/or apoptotic genes in inflammatory bowel disease, making this protein an attractive target for therapeutic intervention.

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NOD2 Modulates Serotonin Transporter and Interacts with TLR2 and TLR4 in Intestinal Epithelial Cells

Cellular Physiology and Biochemistry ◽

10.1159/000490218 ◽

2018 ◽

Vol 47 (3) ◽

pp. 1217-1229 ◽

Cited By ~ 3

Author(s):

Elena Layunta ◽

Eva Latorre ◽

Raquel Forcén ◽

Laura Grasa ◽

Marta Castro ◽

...

Keyword(s):

Innate Immunity ◽

Serotonin Transporter ◽

Intestinal Tract ◽

Inhibitory Effect ◽

Serotonergic System ◽

Epithelial Cell Line ◽

Intestinal Epithelial ◽

Bowel Diseases ◽

Molecular Expression ◽

Sert Activity

Background/Aims: Serotonin (5-HT) is a chief modulator of intestinal activity. The effects of 5-HT depend on its extracellular availability, which is mainly controlled by serotonin transporter (SERT), expressed in enterocytes. On the other hand, innate immunity, mediated by Toll-like receptors (TLRs) and nucleotide oligomerization domain (NOD)-like receptors (NLRs), is known to control intestinal microbiota and maintain intestinal homeostasis. The dysregulation of the intestinal serotonergic system and innate immunity has been observed in inflammatory bowel diseases (IBD), the incidence of which has severely increased all over the world. The aim of the present study, therefore, was to analyze the effect of NOD2 on intestinal SERT activity and expression, as well as to study the crosstalk of NOD2 with TLR2 and TLR4. Methods: Intestinal epithelial cell line Caco-2/TC7 was used to analyze SERT activity and SERT, NOD2, TLR2 and TLR4 molecular expression by real-time PCR and western blotting. Moreover, intestinal tract (ileum and colon) from mice deficient in TLR2, TLR4 or TLR2/4 receptors was used to test the interdependence of NOD2 with these TLR receptors. Results: NOD2 activation inhibits SERT activity in Caco-2/TC7 cells, mainly due to the decrement of SERT molecular expression, with RIP2/RICK being the intracellular pathway involved in this effect. This inhibitory effect on SERT would yield an increment of extracellular 5-HT availability. In this sense, 5-HT strongly inhibits NOD2 expression. In addition, NOD2 showed greater interdependence with TLR2 than with TLR4. Indeed, NOD2 expression significantly increased in both cells treated with TLR2 agonists and the intestinal tract of Tlr2-/- mice. Conclusions: It may be inferred from our data that NOD2 could play a role in intestinal pathophysiology not only through its inherent innate immune role but also due to its interaction with other receptors as TLR2 and the modulation of the intestinal serotonergic system decreasing SERT activity and expression.

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Protective Role of Natural and Semi-Synthetic Tocopherols on TNFα-Induced ROS Production and ICAM-1 and Cl-2 Expression in HT29 Intestinal Epithelial Cells

Antioxidants ◽

10.3390/antiox10020160 ◽

2021 ◽

Vol 10 (2) ◽

pp. 160

Author(s):

Vladana Domazetovic ◽

Irene Falsetti ◽

Caterina Viglianisi ◽

Kristian Vasa ◽

Cinzia Aurilia ◽

...

Keyword(s):

Oxidative Stress ◽

Antioxidant Properties ◽

Intestinal Barrier ◽

Protective Role ◽

Intercellular Adhesion Molecule ◽

Intestinal Epithelial ◽

Intercellular Adhesion Molecule 1 ◽

Beneficial Effects ◽

Bowel Diseases

Vitamin E, a fat-soluble compound, possesses both antioxidant and non-antioxidant properties. In this study we evaluated, in intestinal HT29 cells, the role of natural tocopherols, α-Toc and δ-Toc, and two semi-synthetic derivatives, namely bis-δ-Toc sulfide (δ-Toc)2S and bis-δ-Toc disulfide (δ-Toc)2S2, on TNFα-induced oxidative stress, and intercellular adhesion molecule-1 (ICAM-1) and claudin-2 (Cl-2) expression. The role of tocopherols was compared to that of N-acetylcysteine (NAC), an antioxidant precursor of glutathione synthesis. The results show that all tocopherol containing derivatives used, prevented TNFα-induced oxidative stress and the increase of ICAM-1 and Cl-2 expression, and that (δ-Toc)2S and (δ-Toc)2S2 are more effective than δ-Toc and α-Toc. The beneficial effects demonstrated were due to tocopherol antioxidant properties, but suppression of TNFα-induced Cl-2 expression seems not only to be related with antioxidant ability. Indeed, while ICAM-1 expression is strongly related to the intracellular redox state, Cl-2 expression is TNFα-up-regulated by both redox and non-redox dependent mechanisms. Since ICAM-1 and Cl-2 increase intestinal bowel diseases, and cause excessive recruitment of immune cells and alteration of the intestinal barrier, natural and, above all, semi-synthetic tocopherols may have a potential role as a therapeutic support against intestinal chronic inflammation, in which TNFα represents an important proinflammatory mediator.

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Tu1198 Intestinal Epithelial Cell Extrusion and Microbial Virulence Are Altered in Pediatric Inflammatory Bowel Diseases

Gastroenterology ◽

10.1016/s0016-5085(14)62819-5 ◽

2014 ◽

Vol 146 (5) ◽

pp. S-781

Author(s):

Deenaz Zaidi ◽

Michael Bording-Jorgenson ◽

Hien Q. Huynh ◽

Yuefei Lou ◽

Julia J. Liu ◽

...

Keyword(s):

Epithelial Cell ◽

Inflammatory Bowel Diseases ◽

Intestinal Epithelial Cell ◽

Intestinal Epithelial ◽

Bowel Diseases ◽

Inflammatory Bowel ◽

Cell Extrusion

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Pulchinenosides from Pulsatilla Chinensis Increase P-Glycoprotein Activity and Induce P-Glycoprotein Expression

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/4861719 ◽

2020 ◽

Vol 2020 ◽

pp. 1-8

Author(s):

Yali Liu ◽

Ling Zhang ◽

Shaofeng Wei ◽

Jinyang Cai ◽

Zhenzhong Zang ◽

...

Keyword(s):

Membrane Vesicles ◽

Pcr Analysis ◽

Mrna Levels ◽

Intestinal Epithelial ◽

Glycoprotein P ◽

P Glycoprotein ◽

Human Intestinal Epithelial Cells ◽

Pulsatilla Chinensis ◽

Dependent Transport ◽

Cytotoxic Studies

Five pulchinenosides (pulchinenoside B3, pulchinenoside BD, pulchinenoside B7, pulchinenoside B10, and pulchinenoside B11) isolated from Pulsatilla chinensis (Bge) Regel saponins extract exhibited strong antitumor activities but poor gastrointestinal absorption properties. The enteric induction of P-glycoprotein (P-gp) is understood to restrict the oral bioavailability of some pharmaceutical compounds and lead to adverse drug reactions. Therefore, the present investigation was intended to delineate the impacts of pulchinenosides on cellular P-gp function and expression using Sf9 membrane vesicles and LS180 cells as a surrogate of human intestinal epithelial cells. Preliminary cytotoxic studies showed that 10 μM was an acceptable concentration for cytotoxicity and antiproliferation studies for all pulchinenosides using the alamarBlue assay. The cell cycle of LS180 cells detected by flow cytometry was not significantly influenced after 48 hours of coincubation with 10 μM of pulchinenosides. In the presence of pulchinenosides, the ATP-dependent transport of N-methyl-quinidine mediated by P-glycoprotein was stimulated significantly. The upregulation of P-glycoprotein and mRNA levels was found by Western blot and real-time PCR analysis in LS180 cells. Parallel changes indicate that all pulchinenosides are exposed to pulchinenosides-mediated transcriptional regulation. In conclusion, pulchinenosides could induce P-glycoprotein expression and directly increase its functional activity.

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Inhibition of polyamine synthesis induces p53 gene expression but not apoptosis

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.4.c946 ◽

1999 ◽

Vol 276 (4) ◽

pp. C946-C954 ◽

Cited By ~ 46

Author(s):

Li Li ◽

Ji Li ◽

Jaladanki N. Rao ◽

Minglin Li ◽

Barbara L. Bass ◽

...

Keyword(s):

Gene Expression ◽

Growth Inhibition ◽

P53 Protein ◽

Specific Inhibitor ◽

P53 Gene ◽

Mrna Levels ◽

Intestinal Epithelial ◽

Polyamine Biosynthesis ◽

Fetal Bovine ◽

P53 Mrna

The nuclear phosphoprotein p53 acts as a transcription factor and is involved in growth inhibition and apoptosis. The present study was designed to examine the effect of decreasing cellular polyamines on p53 gene expression and apoptosis in small intestinal epithelial (IEC-6) cells. Cells were grown in DMEM containing 5% dialyzed fetal bovine serum in the presence or absence of α-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, for 4, 6, and 12 days. The cellular polyamines putrescine, spermidine, and spermine in DFMO-treated cells decreased dramatically at 4 days and remained depleted thereafter. Polyamine depletion by DFMO was accompanied by a significant increase in expression of the p53 gene. The p53 mRNA levels increased 4 days after exposure to DFMO, and the maximum increases occurred at 6 and 12 days after exposure. Increased levels of p53 mRNA in DFMO-treated cells were paralleled by increases in p53 protein. Polyamines given together with DFMO completely prevented increased expression of the p53 gene. Increased expression of the p53 gene in DFMO-treated cells was associated with a significant increase in G1 phase growth arrest. In contrast, no features of programmmed cell death were identified after polyamine depletion: no internucleosomal DNA fragmentation was observed, and no morphological features of apoptosis were evident in cells exposed to DFMO for 4, 6, and 12 days. These results indicate that 1) decreasing cellular polyamines increases expression of the p53 gene and 2) activation of p53 gene expression after polyamine depletion does not induce apoptosis in intestinal crypt cells. These findings suggest that increased expression of the p53 gene may play an important role in growth inhibition caused by polyamine depletion.

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Co-activation of synovial fibroblasts by laminin-111 and transforming growth factor-β induces expression of matrix metalloproteinases 3 and 10 independently of nuclear factor-κB

Annals of the Rheumatic Diseases ◽

10.1136/ard.2007.073809 ◽

2007 ◽

Vol 67 (4) ◽

pp. 559-562 ◽

Cited By ~ 4

Author(s):

K Warstat ◽

T Pap ◽

G Klein ◽

S Gay ◽

W K Aicher

Keyword(s):

Growth Factor ◽

Transforming Growth Factor ◽

Synovial Fibroblasts ◽

Transforming Growth Factor Β ◽

Nuclear Factor Κb ◽

Mrna Levels ◽

Nuclear Factor ◽

The Matrix ◽

Specific Tissue ◽

Significant Expression

We showed previously that the attachment of synovial fibroblasts to laminin (LM)-111 in the presence of transforming growth factor-β induces significant expression of the matrix metalloproteinase (MMP)-3. Here we go on to investigate the regulation of additional MMPs and their specific tissue inhibitors of matrix proteases (TIMPs). Changes in steady-state mRNA levels encoding TIMPs and MMPs were investigated by quantitative reverse transcription–polymerase chain reaction. Production of MMPs was monitored by a multiplexed immunoarray. Signal transduction pathways were studied by immunoblotting. Attachment of synovial fibroblasts to LM-111 in the presence of transforming growth factor-β induced significant increases in MMP-3 mRNA (12.35-fold, p<0.001) and protein (mean 62 ng/ml, sixfold, p<0.008) and in expression of MMP-10 mRNA (11.68-fold, p<0.05) and protein (54 ng/ml, 20-fold, p⩾0.02). All other TIMPs and MMPs investigated failed to show this LM-111-facilitated transforming growth factor-β response. No phosphorylation of nuclear factor-κB was observed. We conclude that co-stimulation of synovial fibroblasts by LM-111 together with transforming growth factor-β suffices to induce significant expression of MMP-3 and MMP-10 by synovial fibroblasts and that this induction is independent of nuclear factor-κB phosphorylation.

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