Cytokines induce HIF-1 DNA binding and the expression of HIF-1-dependent genes in cultured rat enterocytes

2003 ◽  
Vol 284 (3) ◽  
pp. G373-G384 ◽  
Author(s):  
Marion Scharte ◽  
Xiaonan Han ◽  
Daniel J. Bertges ◽  
Mitchell P. Fink ◽  
Russell L. Delude
Keyword(s):  
Dna Binding ◽  
Epithelial Cells ◽  
Hypoxia Inducible Factor ◽  
Adaptation To Hypoxia ◽  
Mrna Levels ◽  
Intestinal Epithelial ◽  
Vegf Mrna ◽  
Hypoxic Conditions ◽  
Steady State Levels ◽  
Aldolase A

Cellular adaptation to hypoxia depends, in part, on the transcription factor hypoxia-inducible factor-1 (HIF-1). Normoxic cells exposed to an inflammatory milieu often manifest phenotypic changes, such as increased glycolysis, that are reminiscent of those observed in hypoxic cells. Accordingly, we investigated the effects of cytomix, a mixture containing IFN-γ, TNF, and IL-1β on the expression of HIF-1-dependent proteins under normoxic and hypoxic conditions. Incubation of intestine-derived epithelial cells (IEC-6) under 1% O2increased HIF-1 DNA binding and expression of aldolase A, enolase-1, and VEGF mRNA. Incubation of normoxic cells with cytomix for 48 h also markedly increased HIF-1 DNA binding and expression of mRNAs for these proteins. Incubation of hypoxic cells with cytomix did not inhibit HIF-1 DNA binding or upregulation of HIF-1-dependent genes in response to hypoxia. Neither cytomix nor hypoxia increased steady-state levels of HIF-1α mRNA. Incubation of IEC-6 cells with cytomix induced nitric oxide (NO·) biosynthesis, which was blocked if the cultures containedl- NG-(1-iminoethyl)lysine hydrochloride (l-NIL). Treatment with l-NIL, however, failed to significantly alter aldolase A, enolase-1, and VEGF mRNA levels in normoxic cytomix-treated cells. Proinflammatory cytokines activate the HIF-1 pathway and increase expression of glycolytic genes in nontransformed rat intestinal epithelial cells, largely through an NO·-independent mechanism.

scholarly journals Hypoxic Environment Promotes Barrier Formation in Human Intestinal Epithelial Cells through Regulation of MicroRNA 320a Expression

2019 ◽  
Vol 39 (14) ◽  
Author(s):  
Stephanie Muenchau ◽  
Rosalie Deutsch ◽  
Ines J. de Castro ◽  
Thomas Hielscher ◽  
Nora Heber ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mirna Expression ◽  
Barrier Function ◽  
Intestinal Epithelial Cells ◽  
Hypoxia Inducible Factor ◽  
Intestinal Epithelial ◽  
Low Oxygen ◽  
Direct Role ◽  
Hypoxic Conditions ◽  
Barrier Formation

ABSTRACT Intestinal epithelial cells (IECs) are exposed to the low-oxygen environment present in the lumen of the gut. These hypoxic conditions on one hand are fundamental for the survival of the commensal microbiota and, on the other hand, favor the formation of a selective semipermeable barrier, allowing IECs to transport essential nutrients/water while keeping the sterile internal compartments separated from the lumen containing commensals. The hypoxia-inducible factor (HIF) complex, which allows cells to respond and adapt to fluctuations in oxygen levels, has been described as a key regulator in maintaining IEC barrier function by regulating their tight junction integrity. In this study, we sought to better evaluate the mechanisms by which low oxygen conditions impact the barrier function of human IECs. By profiling miRNA expression in IECs under hypoxia, we identified microRNA 320a (miRNA-320a) as a novel barrier formation regulator. Using pharmacological inhibitors and short hairpin RNA-mediated silencing, we could demonstrate that expression of this microRNA (miRNA) was HIF dependent. Importantly, using overexpression and knockdown approaches of miRNA-320a, we could confirm its direct role in the regulation of barrier function in human IECs. These results reveal an important link between miRNA expression and barrier integrity, providing a novel insight into mechanisms of hypoxia-driven epithelial homeostasis.

scholarly journals Intrauterine Exposure to Cadmium Reduces HIF-1 DNA-Binding Ability in Rat Fetal Kidneys

Toxics ◽  
2018 ◽  
Vol 6 (3) ◽  
pp. 53 ◽  
Author(s):  
Tania Jacobo-Estrada ◽  
Mariana Cardenas-Gonzalez ◽  
Mitzi Santoyo-Sánchez ◽  
Frank Thevenod ◽  
Olivier Barbier
Keyword(s):  
Dna Binding ◽  
Kidney Development ◽  
Protein Extraction ◽  
Hypoxia Inducible Factor ◽  
Isotonic Saline ◽  
Mrna Levels ◽  
Vegf Mrna ◽  
Binding Ability ◽  
Protein Levels ◽  
Cd Exposure

During embryonic development, some hypoxia occurs due to incipient vascularization. Under hypoxic conditions, gene expression is mainly controlled by hypoxia-inducible factor 1 (HIF-1). The activity of this transcription factor can be altered by the exposure to a variety of compounds; among them is cadmium (Cd), a nephrotoxic heavy metal capable of crossing the placenta and reaching fetal kidneys. The goal of the study was to determine Cd effects on HIF-1 on embryonic kidneys. Pregnant Wistar rats were exposed to a mist of isotonic saline solution or CdCl2 (DDel = 1.48 mg Cd/kg/day), from gestational day (GD) 8 to 20. Embryonic kidneys were obtained on GD 21 for RNA and protein extraction. Results show that Cd exposure had no effect on HIF-1α and prolyl hydroxylase 2 protein levels, but it reduced HIF-1 DNA-binding ability, which was confirmed by a decrease in vascular endothelial growth factor (VEGF) mRNA levels. In contrast, the protein levels of VEGF were not changed, which suggests the activation of additional regulatory mechanisms of VEGF protein expression to ensure proper kidney development. In conclusion, Cd exposure decreases HIF-1-binding activity, posing a risk on renal fetal development.

scholarly journals Hypoxic environment promotes barrier formation in human intestinal epithelial cells through regulation of miRNA-320a expression

2018 ◽  
Author(s):  
Stephanie Muenchau ◽  
Rosalie Deutsch ◽  
Thomas Hielscher ◽  
Nora Heber ◽  
Beate Niesler ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mirna Expression ◽  
Barrier Function ◽  
Intestinal Epithelial Cells ◽  
Hypoxia Inducible Factor ◽  
Intestinal Epithelial ◽  
Low Oxygen ◽  
Direct Role ◽  
Hypoxic Conditions ◽  
Barrier Formation

AbstractIntestinal epithelial cells (IECs) are exposed to the low-oxygen environment present in the lumen of the gut. These hypoxic conditions are on one hand fundamental for the survival of the commensal microbiota, and on the other hand, favor the formation of a selective semipermeable barrier allowing IECs to transport essential nutrients/water while keeping the sterile internal compartments separated from the lumen containing commensals. The hypoxia-inducible factor (HIF) complex, which allows cells to respond and adapt to fluctuations in oxygen levels, has been described as a key regulator in maintaining IEC barrier function by regulating their tight junction integrity. In this study, we sought to better evaluate the mechanisms by which low oxygen conditions impact the barrier function of human IECs. By profiling miRNA expression in IECs under hypoxia, we identified miRNA-320a as a novel barrier formation regulator. Using pharmacological inhibitors and short hairpin RNA-mediated silencing we could demonstrate that expression of this miRNA was HIF-dependent. Importantly, using over-expression and knock-down approaches of miRNA-320a we could confirm its direct role in the regulation of barrier functions in human IECs. These results reveal an important link between miRNA expression and barrier integrity, providing a novel insight into mechanisms of hypoxia-driven epithelial homeostasis.

BUTYRATE SUPPRESSES HYPOXIA-INDUCIBLE FACTOR-1 ACTIVITY IN INTESTINAL EPITHELIAL CELLS UNDER HYPOXIC CONDITIONS

Shock ◽  
2004 ◽  
Vol 22 (5) ◽  
pp. 446-452 ◽  
Author(s):  
Keita Miki ◽  
Naoki Unno ◽  
Toshi Nagata ◽  
Masato Uchijima ◽  
Hiroyuki Konno ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Hypoxia Inducible Factor ◽  
Intestinal Epithelial ◽  
Hypoxia Inducible Factor 1 ◽  
Hypoxic Conditions

Comparison of intestinal folate carrier clone expressed in IEC-6 cells and in Xenopusoocytes

1998 ◽  
Vol 274 (1) ◽  
pp. C289-C294 ◽  
Author(s):  
Chandira K. Kumar ◽  
Toai T. Nguyen ◽  
Francis B. Gonzales ◽  
Hamid M. Said
Keyword(s):  
Folic Acid ◽  
Epithelial Cells ◽  
Cdna Clone ◽  
Xenopus Oocytes ◽  
Intestinal Epithelial Cells ◽  
Maximal Velocity ◽  
Mrna Levels ◽  
Acidic Ph ◽  
Intestinal Epithelial ◽  
Folate Transport

We recently identified a cDNA clone from mouse small intestine, which appears to be involved in folate transport when expressed in Xenopus oocytes. The open reading frame of this clone is identical to that of the reduced folate carrier (RFC) (K. H. Dixon, B. C. Lanpher, J. Chiu, K. Kelley, and K. H. Cowan. J. Biol. Chem. 269: 17–20, 1994). The characteristics of this cDNA clone [previously referred to as intestinal folate carrier 1 (IFC-1)] expressed in Xenopus oocytes, however, were found to be different from the characteristics of folate transport in native small intestinal epithelial cells. To further study these differences, we determined the characteristics of RFC when expressed in an intestinal epithelial cell line, IEC-6, and compared the findings to its characteristics when expressed in Xenopus oocytes. RFC was stably transfected into IEC-6 cells by electroporation; its cRNA was microinjected into Xenopus oocytes. Northern blot analysis of poly(A)+RNA from IEC-6 cells stably transfected with RFC cDNA (IEC-6/RFC) showed a twofold increase in RFC mRNA levels over controls. Similarly, uptake of folic acid and 5-methyltetrahydrofolate (5-MTHF) by IEC-6/RFC was found to be fourfold higher than uptake in control sublines. This increase in folic acid and 5-MTHF uptake was inhibited by treating IEC-6/RFC cells with cholesterol-modified antisense DNA oligonucleotides. The increase in uptake was found to be mainly mediated through an increase in the maximal velocity ( V max) of the uptake process [the apparent Michaelis-Menten constant ( K m) also changed (range was 0.31 to 1.56 μM), but no specific trend was seen]. In both IEC-6/RFC and control sublines, the uptake of both folic acid and 5-MTHF displayed 1) pH dependency, with a higher uptake at acidic pH 5.5 compared with pH 7.5, and 2) inhibition to the same extent by both reduced and oxidized folate derivatives. These characteristics are very similar to those seen in native intestinal epithelial cells. In contrast, RFC expressed in Xenopus oocytes showed 1) higher uptake at neutral and alkaline pH 7.5 compared with acidic pH 5.5 and 2) higher sensitivity to reduced compared with oxidized folate derivatives. Results of these studies demonstrate that the characteristics of RFC vary depending on the cell system in which it is expressed. Furthermore, the results may suggest the involvement of cell- or tissue-specific posttranslational modification(s) and/or the existence of an auxiliary protein that may account for the differences in the characteristics of the intestinal RFC when expressed in Xenopus oocytes compared with when expressed in intestinal epithelial cells.

scholarly journals Mitochondrial gene expression in small intestinal epithelial cells

1995 ◽  
Vol 308 (2) ◽  
pp. 665-671 ◽  
Author(s):  
T P Mayall ◽  
I Bjarnason ◽  
U Y Khoo ◽  
T J Peters ◽  
A J S Macpherson
Keyword(s):  
Epithelial Cells ◽  
Cytochrome Oxidase ◽  
Cytochrome B ◽  
Intestinal Epithelial Cells ◽  
Mitochondrial Gene ◽  
Cytochrome Oxidase I ◽  
Mrna Levels ◽  
Intestinal Epithelial ◽  
Mitochondrial Transcription Factor ◽  
Small Intestinal

Most mitochondrial genes are transcribed as a single large transcript from the heavy strand of mitochondrial DNA, and are subsequently processed into the proximal mitochondrial (mt) 12 S and 16 S rRNAs, and the more distal tRNAs and mRNAs. We have shown that in intestinal epithelial biopsies the steady-state levels of mt 12 S and 16 S rRNA are an order of magnitude greater than those of mt mRNAs. Fractionation of rat small intestinal epithelial cells on the basis of their maturity has shown that the greatest ratios of 12 S mt rRNA/cytochrome b mt mRNA or 12 S mt rRNA/cytochrome oxidase I mt mRNA are found in the surface mature enterocytes, with a progressive decrease towards the crypt immature enteroblasts. Cytochrome b and cytochrome oxidase I mt mRNA levels are relatively uniform along the crypt-villus axis, but fractionation experiments showed increased levels in the crypt base. The levels of human mitochondrial transcription factor A are also greater in immature crypt enteroblasts compared with mature villus enterocytes. These results show that the relative levels of mt rRNA and mRNA are distinctly regulated in intestinal epithelial cells according to the crypt-villus position and differentiation status of the cells, and that there are higher mt mRNA and mt TFA levels in the crypts, consistent with increased transcriptional activity during mitochondrial biogenesis in the immature enteroblasts.

scholarly journals Inhibitor of DNA Binding 1 Is Induced during Kidney Ischemia-Reperfusion and Is Critical for the Induction of Hypoxia-Inducible Factor-1α

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Dan Wen ◽  
Yan-Fang Zou ◽  
Yao-Hui Gao ◽  
Qian Zhao ◽  
Yin-Yin Xie ◽  
...  
Keyword(s):  
Dna Binding ◽  
Ischemia Reperfusion ◽  
Hypoxia Inducible Factor ◽  
Kidney Injury ◽  
Mrna Levels ◽  
Hypoxia Inducible Factor 1 ◽  
Renal Tubular ◽  
Inhibitor Of Dna Binding

In this study, rat models of acute kidney injury (AKI) induced by renal ischemia-reperfusion (I/R) and HK-2 cell models of hypoxia-reoxygenation (H/R) were established to investigate the expression of inhibitor of DNA binding 1 (ID1) in AKI, and the regulation relationship between ID1 and hypoxia-inducible factor 1 alpha (HIF-1α). Through western blot, quantitative real-time PCR, immunohistochemistry, and other experiment methods, the induction of ID1 after renal I/R in vivo was observed, which was expressed mainly in renal tubular epithelial cells (TECs). ID1 expression was upregulated in in vitro H/R models at both the protein and mRNA levels. Via RNAi, it was found that ID1 induction was inhibited with silencing of HIF-1α. Moreover, the suppression of ID1 mRNA expression could lead to decreased expression and transcription of HIF-1αduring hypoxia and reoxygenation. In addition, it was demonstrated that both ID1 and HIF-1αcan regulate the transcription of twist. This study demonstrated that ID1 is induced in renal TECs during I/R and can regulate the transcription and expression of HIF-1α.

scholarly journals Alteration in Inflammatory Responses and Cytochrome P450 Expression of Porcine Jejunal Cells by Drinking Water Supplements

2019 ◽  
Vol 2019 ◽  
pp. 1-6 ◽  
Author(s):  
Orsolya Palócz ◽  
Géza Szita ◽  
György Csikó
Keyword(s):  
Gene Expression ◽  
Drinking Water ◽  
Cytochrome P450 ◽  
Epithelial Cells ◽  
Fulvic Acid ◽  
Intestinal Epithelial Cells ◽  
Feed Additives ◽  
Feed Additive ◽  
Mrna Levels ◽  
Intestinal Epithelial

The intestinal epithelium is the first determining barrier to the drugs administered per os. Cytochrome P450 (CYP) enzymes are substantial in the initial step of xenobiotic metabolism; therefore, intestinal CYP enzyme activities could be an important influencing factor of the oral utilization of xenobiotic substances. In this study, the effect of four drinking water supplements on CYP mRNA levels of porcine intestinal epithelial cells was examined. Further goal of the study is to describe the effect of these feed additives on the proinflammatory response of the LPS-treated enterocytes. The nontransformed porcine intestinal epithelial cells (IPEC-J2) were grown on six-well polyester membrane inserts. Cell cultures were treated with LPS (10 μg/ml), β-glucan (5 and 50 μg/ml), sanguinarine-containing additive (5 and 50 μg/ml), drinking water acidifier (0.1 and 1 μl/ml), and fulvic acid (25 and 250 μg/ml) for 1 hour. Cells were washed with culture medium and incubated for additional 1 h before total RNA isolation. IL-6, IL-8, TNF-α, HSP70, CYP1A1, CYP1A2, and CYP3A29 mRNA levels were measured. The LPS treatment upregulated the gene expression of IL-8 and TNF-α. The relative gene expression of IL-6 remained unchanged and TNF-α and HSP70 were downregulated after the treatment with each feed additive. CYP1A1 and CYP1A2 expressions increased after sanguinarine-containing solution, fulvic acid, and drinking water acidifier treatment. None of the treatments changed the gene expression of CYP3A29, responsible for the metabolism of the majority of drug substances used in swine industry. The feed additive substances inhibited the expression of proinflammatory mediators HSP70 and TNF-α; however, β-glucan and fulvic acid elevated the production of the chemokine IL-8 mRNA in endotoxin-treated enterocytes. All acidic supplements increased the expression of CYP1A1 gene; their constituents may serve as a ligand of CYP1A1 nuclear receptors.

scholarly journals The HIF target ATG9A is essential for epithelial barrier function and tight junction biogenesis

2020 ◽  
Vol 31 (20) ◽  
pp. 2249-2258
Author(s):  
Alexander S. Dowdell ◽  
Ian M. Cartwright ◽  
Matthew S. Goldberg ◽  
Rachael Kostelecky ◽  
Tyler Ross ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Tight Junction ◽  
Barrier Function ◽  
Intestinal Epithelial Cells ◽  
Hypoxia Inducible Factor ◽  
Epithelial Barrier ◽  
Adaptation To Hypoxia ◽  
Intestinal Epithelial ◽  
Hypoxia Response ◽  
Epithelial Barrier Function

The transcription factor hypoxia-inducible factor (HIF) mediates adaptation to hypoxia. We found that HIF regulates the autophagy protein ATG9A in intestinal epithelial cells. Subsequent knockdown of ATG9A resulted in tight junction mislocalization and cytoskeletal defects. These results suggest a link among the hypoxia response, autophagy, and junctional biogenesis.

The human HIF (hypoxia-inducible factor)-3α gene is a HIF-1 target gene and may modulate hypoxic gene induction

2009 ◽  
Vol 424 (1) ◽  
pp. 143-151 ◽  
Author(s):  
Tetsuhiro Tanaka ◽  
Michael Wiesener ◽  
Wanja Bernhardt ◽  
Kai-Uwe Eckardt ◽  
Christina Warnecke
Keyword(s):  
Target Gene ◽  
Splice Variants ◽  
Lysyl Oxidase ◽  
Hypoxia Inducible Factor ◽  
Gene Induction ◽  
Adaptation To Hypoxia ◽  
Transcriptional Level ◽  
Dependent Manner ◽  
Hypoxic Conditions ◽  
Chemical Hypoxia

HIF (hypoxia-inducible factor)-3α is the third member of the HIF transcription factor family. Whereas HIF-1α and -2α play critical roles in the cellular and systemic adaptation to hypoxia, little is known about the regulation and function of HIF-3α. At least five different splice variants may be expressed from the human HIF-3α locus that are suggested to exert primarily negative regulatory effects on hypoxic gene induction. In the present paper, we report that hypoxia induces the human HIF-3α gene at the transcriptional level in a HIF-1-dependent manner. HIF-3α2 and HIF-3α4 transcripts, the HIF-3α splice variants expressed in Caki-1 renal carcinoma cells, rapidly increased after exposure to hypoxia or chemical hypoxia mimetics. siRNA (small interfering RNA)-mediated HIF-α knockdown demonstrated that HIF-3α is a specific target gene of HIF-1α, but is not affected by HIF-2α knockdown. In contrast with HIF-1α and HIF-2α, HIF-3α is not regulated at the level of protein stability. HIF-3α protein could be detected under normoxia in the cytoplasm and nuclei, but increased under hypoxic conditions. Promoter analyses and chromatin immunoprecipitation experiments localized a functional hypoxia-responsive element 5′ to the transcriptional start of HIF-3α2. siRNA-mediated knockdown of HIF-3α increased transactivation of a HIF-driven reporter construct and mRNA expression of lysyl oxidase. Immunohistochemistry revealed an overlap of HIF-1α-positive and HIF-3α-positive areas in human renal cell carcinomas. These findings shed light on a novel aspect of HIF-3α as a HIF-1 target gene and point to a possible role as a modulator of hypoxic gene induction.

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