scholarly journals Induction of Gastrin Expression in Gastrointestinal Cells by Hypoxia or Cobalt Is Independent of Hypoxia-Inducible Factor (HIF)

Endocrinology ◽  
2012 ◽  
Vol 153 (7) ◽  
pp. 3006-3016 ◽  
Author(s):  
Lin Xiao ◽  
Suzana Kovac ◽  
Mike Chang ◽  
Arthur Shulkes ◽  
Graham S. Baldwin ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Mutational Analysis ◽  
Hypoxia Inducible Factor ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Human Gastric Cancer ◽  
Low Oxygen ◽  
Ags Cells ◽  
Cholecystokinin Receptor ◽  
Hypoxia Inducible Factor 1

Gastrin and its precursors have been shown to promote mitogenesis and angiogenesis in gastrointestinal tumors. Hypoxia stimulates tumor growth, but its effect on gastrin gene regulation has not been examined in detail. Here we have investigated the effect of hypoxia on the transcription of the gastrin gene in human gastric cancer (AGS) cells. Gastrin mRNA was measured by real-time PCR, gastrin peptides were measured by RIA, and gastrin promoter activity was measured by dual-luciferase reporter assay. Exposure to a low oxygen concentration (1%) increased gastrin mRNA concentrations in wild-type AGS cells (AGS) and in AGS cells overexpressing the gastrin receptor (AGS-cholecystokinin receptor 2) by 2.1 ± 0.4- and 4.1 ± 0.3-fold (P < 0.05), respectively. The hypoxia mimetic, cobalt chloride (300 μM), increased gastrin promoter activity in AGS cells by 2.4 ± 0.3-fold (P < 0.05), and in AGS-cholecystokinin receptor 2 cells by 4.0 ± 0.3-fold (P < 0.05), respectively. The observations that either deletion from the gastrin promoter of the putative binding sites for the transcription factor hypoxia-inducible factor 1 (HIF-1) or knockdown of either the HIF-1α or HIF-1β subunit did not affect gastrin promoter inducibility under hypoxia indicated that the hypoxic activation of the gastrin gene is likely HIF independent. Mutational analysis of previously identified Sp1 regulatory elements in the gastrin promoter also failed to abrogate the induction of promoter activity by hypoxia. The observations that hypoxia up-regulates the gastrin gene in AGS cells by HIF-independent mechanisms, and that this effect is enhanced by the presence of gastrin receptors, provide potential targets for gastrointestinal cancer therapy.

scholarly journals Hypoxia-Inducible Factor-1 Transactivates Transforming Growth Factor-β3 in Trophoblast

Endocrinology ◽  
2004 ◽  
Vol 145 (9) ◽  
pp. 4113-4118 ◽  
Author(s):  
Hirotaka Nishi ◽  
Toshihide Nakada ◽  
Mitsuyasu Hokamura ◽  
Yumi Osakabe ◽  
Osamu Itokazu ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Transforming Growth Factor ◽  
Hypoxia Inducible Factor ◽  
First Trimester ◽  
Vital Role ◽  
Low Oxygen ◽  
Mobility Shift ◽  
Trophoblast Differentiation ◽  
Hypoxia Inducible Factor 1 ◽  
Electrophoretic Mobility Shift Assays

Abstract Hypoxia occurs during the development of placenta in the first trimester and is implicated in trophoblast differentiation. Intervillous blood flow increases after 10 wk of gestation and results in exposure of trophoblast cells to oxygen. Before this time, low oxygen appears to prevent trophoblast differentiation toward an invasive phenotype. The oxygen-regulated early events of trophoblast differentiation are mediated by TGF-β3. TGF-β3 plays a vital role in trophoblast differentiation, and its overexpression can be found in preeclamptic placenta. We sought to determine the mechanism of TGF-β3 expression through hypoxia-inducible factor (HIF)-1. We show that HIF-1α and TGF-β3 are overexpressed in preeclamptic placenta. Hypoxia not only transactivates the TGF-β3 promoter activity but also enhances endogenous TGF-β3 expression. Using the TGF-β3 promoter deletion mutants, we show that the region between −90 and −60, which contains a putative HIF-1 consensus motif, is crucial for HIF-1-mediated transactivation. Electrophoretic mobility shift assays show that HIF-1 binds to the oligonucleotide containing the HIF-1 motif. Also, introduction of an antisense oligonucleotide for HIF-1 diminishes TGF-β3 expression during hypoxia, indicating that the up-regulation of TGF-β3 by hypoxia is mediated through HIF-1. Our results provide evidence that regulation of TGF-β3 promoter activity by HIF-1 represents a mechanism for trophoblast differentiation during hypoxia.

scholarly journals Oxygen impairs oligodendroglial development via oxidative stress and reduced expression of HIF-1α

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Christina Brill ◽  
Till Scheuer ◽  
Christoph Bührer ◽  
Stefanie Endesfelder ◽  
Thomas Schmitz
Keyword(s):  
Oxidative Stress ◽  
Hypoxia Inducible Factor ◽  
Precursor Cell ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Low Oxygen ◽  
Hypoxia Inducible Factor 1 ◽  
Reporter Assays ◽  
Induced Changes ◽  
Cells Cultured

Abstract The premature increase of oxygen tension may contribute to oligodendroglial precursor cell (OPC) damage in preterm infants. Fetal OPCs are exposed to low oxygen tissue tensions not matched when cells are cultured in room air. Maturation (A2B5, O4, O1, MBP, CNP, arborization), oxidative stress (nitrotyrosine Western blot, NRF2 and SOD2 expression), apoptosis (TUNEL), proliferation (Ki67), and expression of transcription factors regulated by Hypoxia-Inducible-Factor-1-alpha (Hif-1α) expressed in OPCs (Olig1, Olig2, Sox9, Sox10) were assessed in rat OPCs and OLN93 cells cultured at 5% O2 and 21% O2. Influences of Hif-1α were investigated by Hif-1α luciferase reporter assays and Hif-1α-knockdown experiments. At 21% O2, cell proliferation was decreased and process arborization of OPCs was reduced. Expression of MBP, CNP, Olig1, Sox9 and Sox10 was lower at 21% O2, while Nrf2, SOD2, nitrotyrosine were increased. Apoptosis was unchanged. Luciferease reporter assay in OLN93 cells indicated increased Hif-1α activity at 5% O2. In OLN93 cells at 5% O2, Hif-1α knockdown decreased the expression of MBP and CNP, similar to that observed at 21% O2. These data indicate that culturing OPCs at 21% O2 negatively affects development and maturation. Both enhanced oxidative stress and reduced expression of Hif-1α-regulated genes contribute to these hyperoxia-induced changes.

scholarly journals Transcriptional Activation of the Decidual/Trophoblast Prolactin-Related Protein Gene1

Endocrinology ◽  
1999 ◽  
Vol 140 (9) ◽  
pp. 4032-4039 ◽  
Author(s):  
Kyle E. Orwig ◽  
Michael J. Soares
Keyword(s):  
Transcriptional Activation ◽  
Promoter Activity ◽  
Mutational Analysis ◽  
Regulatory Element ◽  
Gene Activation ◽  
Cell Formation ◽  
Regulatory Elements ◽  
Related Protein ◽  
Trophoblast Cells ◽  
Decidual Cells

Abstract The decidual/trophoblast PRL-related protein (d/tPRP) is dually expressed by decidual and trophoblast cells during pregnancy. We have characterized the proximal d/tPRP promoter responsible for directing d/tPRP expression in decidual and trophoblast cells. We have demonstrated that the proximal 93 bp of d/tPRP 5′-flanking DNA are sufficient to direct luciferase gene expression in primary decidual and Rcho-1 trophoblast cells, but not in fibroblast, undifferentiated uterine stromal cells or trophoblast cells of a labyrinthine lineage. The 93-bp d/tPRP promoter was also sufficient to direct differentiation-dependent expression in trophoblast giant cells. Mutational analysis demonstrated the differential importance of activating protein-1 and Ets regulatory elements (located within the proximal 93 bp of d/tPRP 5′-flanking DNA) for activation of the d/tPRP promoter in decidual vs. trophoblast cells. Disruption of the activating protein-1 regulatory element inhibited d/tPRP promoter activity by more than 95% in decidual cells, and approximately 80% trophoblast cells. Disruption of the Ets regulatory element reduced d/tPRP promoter activity by approximately 50% in decidual cells, while inactivating the d/tPRP promoter in trophoblast cells. Protein interactions with the trophoblast Ets regulatory element were shown to be cell type specific and to change during trophoblast giant cell formation. In conclusion, a 93-bp region of the d/tPRP promoter is shown to contain regulatory elements sufficient for gene activation in decidual and trophoblast cells.

scholarly journals Transcriptional Regulation of BK Virus by Nuclear Factor of Activated T Cells

2009 ◽  
Vol 84 (4) ◽  
pp. 1722-1730 ◽  
Author(s):  
Joslynn A. Jordan ◽  
Kate Manley ◽  
Aisling S. Dugan ◽  
Bethany A. O'Hara ◽  
Walter J. Atwood
Keyword(s):  
T Cells ◽  
Promoter Activity ◽  
Mutational Analysis ◽  
Adult Population ◽  
Bk Virus ◽  
Human Polyomavirus ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Transplant Patients

ABSTRACT The human polyomavirus BK virus (BKV) is a common virus for which 80 to 90% of the adult population is seropositive. BKV reactivation in immunosuppressed patients or renal transplant patients is the primary cause of polyomavirus-associated nephropathy (PVN). Using the Dunlop strain of BKV, we found that nuclear factor of activated T cells (NFAT) plays an important regulatory role in BKV infection. Luciferase reporter assays and chromatin immunoprecipitation assays demonstrated that NFAT4 bound to the viral promoter and regulated viral transcription and infection. The mutational analysis of the NFAT binding sites demonstrated complex functional interactions between NFAT, c-fos, c-jun, and the p65 subunit of NF-κB that together influence promoter activity and viral growth. These data indicate that NFAT is required for BKV infection and is involved in a complex regulatory network that both positively and negatively influences promoter activity and viral infection.

scholarly journals Sp1-Mediated Transcription of the Werner Helicase Gene Is Modulated by Rb and p53

1998 ◽  
Vol 18 (11) ◽  
pp. 6191-6200 ◽  
Author(s):  
Yukako Yamabe ◽  
Akira Shimamoto ◽  
Makoto Goto ◽  
Jun Yokota ◽  
Minoru Sugawara ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Transcriptional Control ◽  
Housekeeping Genes ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Control Element ◽  
Mrna Levels

ABSTRACT The regulation of Werner’s syndrome gene (WRN) expression was studied by characterizing the cis-regulatory elements in the promoter region and the trans-activating factors that bind to them. First, we defined the transcription initiation sites and the sequence of the 5′ upstream region (2.8 kb) ofWRN that contains a number of cis-regulatory elements, including 7 Sp1, 9 retinoblastoma control element (RCE), and 14 AP2 motifs. A region consisting of nucleotides −67 to +160 was identified as the principal promoter of WRN by reporter gene assays in HeLa cells, using a series of WRNpromoter-luciferase reporter (WRN-Luc) plasmids that contained the 5′-truncated or mutated WRN upstream regions. In particular, two Sp1 elements proximal to the transcription initiation site are indispensable for WRN promoter activity and bind specifically to Sp1 proteins. The RCE enhances WRN promoter activity. Coexpression of the WRN-Luc plasmids with various dosages of plasmids expressing Rb or p53 in Saos2 cells lacking active Rb and p53 proteins showed that the introduced Rb upregulates WRN promoter activity a maximum of 2.5-fold, while p53 downregulates it a maximum of 7-fold, both dose dependently. Consistently, the overexpressed Rb and p53 proteins also affected the endogenous WRN mRNA levels in Saos2 cells, resulting in an increase with Rb and a decrease with p53. These findings suggest that WRN expression, like that of other housekeeping genes, is directed mainly by the Sp1 transcriptional control system but is also further modulated by transcription factors, including Rb and p53, that are implicated in the cell cycle, cell senescence, and genomic instability.

scholarly journals A hypoxia-independent up-regulation of hypoxia-inducible factor-1 by AKT contributes to angiogenesis in human gastric cancer

2007 ◽  
Vol 29 (1) ◽  
pp. 44-51 ◽  
Author(s):  
B. L. Lee ◽  
W. H. Kim ◽  
J. Jung ◽  
S. J. Cho ◽  
J.-W. Park ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Hypoxia Inducible Factor ◽  
Human Gastric Cancer ◽  
Hypoxia Inducible Factor 1

scholarly journals Isoproterenol and cAMP regulation of the human brain natriuretic peptide gene involves Src and Rac

2000 ◽  
Vol 278 (6) ◽  
pp. E1115-E1123 ◽  
Author(s):  
Quan He ◽  
Guiyun Wu ◽  
Margot C. Lapointe
Keyword(s):  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Promoter Activity ◽  
Kinase Inhibitor ◽  
Regulatory Elements ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Dominant Negative Mutant ◽  
Luciferase Reporter Gene ◽  
Src Tyrosine Kinase

Brain natriuretic peptide (BNP) gene expression and chronic activation of the sympathetic nervous system are characteristics of the development of heart failure. We studied the role of the β-adrenergic signaling pathway in regulation of the human BNP (hBNP) promoter. An hBNP promoter (−1818 to +100) coupled to a luciferase reporter gene was transferred into neonatal cardiac myocytes, and luciferase activity was measured as an index of promoter activity. Isoproterenol (ISO), forskolin, and cAMP stimulated the promoter, and the β2-antagonist ICI 118,551 abrogated the effect of ISO. In contrast, the protein kinase A (PKA) inhibitor H-89 failed to block the action of cAMP and ISO. Pertussis toxin (PT), which inactivates Gαi, inhibited ISO- and cAMP-stimulated hBNP promoter activity. The Src tyrosine kinase inhibitor PP1 and a dominant-negative mutant of the small G protein Rac also abolished the effect of ISO and cAMP. Finally, we studied the involvement of M-CAT-like binding sites in basal and inducible regulation of the hBNP promoter. Mutation of these elements decreased basal and cAMP-induced activity. These data suggest that β-adrenergic regulation of hBNP is PKA independent, involves a Gαi-activated pathway, and targets regulatory elements in the proximal BNP promoter.

scholarly journals Hypoxic activation of the atrial natriuretic peptide gene promoter through direct and indirect actions of hypoxia-inducible factor-1

2003 ◽  
Vol 370 (1) ◽  
pp. 149-157 ◽  
Author(s):  
Yang-Sook CHUN ◽  
Ju-Yeon HYUN ◽  
Yong-Geun KWAK ◽  
In-San KIM ◽  
Chan-Hyung KIM ◽  
...  
Keyword(s):  
Atrial Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Promoter Activity ◽  
Hypoxia Inducible Factor ◽  
Gene Promoter ◽  
Ventricular Myocardium ◽  
Dominant Negative ◽  
Hypoxia Inducible Factor 1 ◽  
Binding Sequence ◽  
Atrial Natriuretic

Atrial natriuretic peptide (ANP) is a cardiac peptide, the transcription of which is up-regulated in the ischaemic ventricle. However, the molecular mechanism of ANP induction is unclear. This study demonstrated that ANP mRNA expression in rat ventricular myocardium is induced in an early phase of ischaemia, preceded by hypoxia-inducible factor-1 (HIF-1) α expression. The ANP gene was also induced by hypoxia or HIF-1 inducers such as CoCl2 and desferrioxamine in H9c2 and neonatal cardiomyocytes. The 2307bp 5′-flanking region of the rat ANP gene was cloned and fused to the luciferase gene. Evidence of the promoter activity was only apparent in the myocytes and was induced by hypoxia and HIF-1 inducers. The overexpression of HIF-1α markedly enhanced ANP promoter activity, and a dominant-negative isoform completely suppressed it. We demonstrated that the promoter regions are essential for hypoxic ANP induction. One promoter region, containing the HIF-1-binding sequence, is regulated directly by HIF-1. The other region is also activated by HIF-1 despite having no HIF-1-binding sequence. These results suggest that HIF-1 enhances the transactivation of the ANP gene in hypoxic myocytes, implying that stimulation of the ANP promoter by HIF-1 may in fact be responsible for the induction of the ANP gene in ischaemic ventricular myocardium.

KLF4 positively regulates human ghrelin expression

2009 ◽  
Vol 420 (3) ◽  
pp. 403-411 ◽  
Author(s):  
Hyo Jung Lee ◽  
Young Mi Kang ◽  
Chang Suk Moon ◽  
Myung Kuk Joe ◽  
Joo Hyun Lim ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Human Gastric Cancer ◽  
Dependent Manner ◽  
Growth Hormone Secretagogue Receptor ◽  
Mobility Shift ◽  
Ags Cells ◽  
Growth Hormone Secretagogue ◽  
Gh Secretion ◽  
Klf4 Expression ◽  
Responsive Element

Ghrelin, an endogenous ligand of the GH (growth hormone) secretagogue receptor, influences many metabolic processes including GH secretion, food intake, energy balance, insulin secretion and adipogenesis. Although ghrelin exhibits a variety of biological functions, the mechanism by which ghrelin expression is regulated is unknown. Ghrelin is expressed in the gastrointestinal tract, predominantly in the stomach, as is KLF4 (Krüppel-like factor 4). Therefore we investigated whether ghrelin expression is associated with KLF4, and found that the tissue distribution of ghrelin corresponded with that of KLF4. Furthermore, treatment with butyrate, an inducer of KLF4 expression, stimulated ghrelin expression, and fasting, which induces ghrelin expression, also increased KLF4 expression, suggesting that ghrelin expression is associated with KLF4. Then, we investigated the effects of KLF4 on the human ghrelin-promoter activity and identified a KLF4-responsive region in the promoter. KLF4 expression specifically stimulated human ghrelin-promoter activity in a dose-dependent manner in human gastric-cancer AGS cells. However, this effect was not seen in response to a mutant KLF4 construct. Transfection studies using mutant constructs containing 5′-deletions in the human ghrelin promoter revealed that the KLF4-responsive element is located between −1228 and −1105. Electrophoretic mobility shift assays using oligonucleotides containing −1165/−1146 revealed the binding of KLF4 to the human ghrelin promoter. Finally, deletion of the −1165/−1146 region abrogated KLF4-induced transactivation of the ghrelin promoter. Collectively, these results indicate that KLF4 positively regulates human ghrelin expression via binding to a KLF-responsive region in the promoter.

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