Regulation of the human biotin transporter hSMVT promoter by KLF-4 and AP-2: confirmation of promoter activity in vivo

2007 ◽  
Vol 292 (4) ◽  
pp. C1305-C1312 ◽  
Author(s):  
Jack C. Reidling ◽  
Hamid M. Said
Keyword(s):  
Epithelial Cell ◽  
Cell Lines ◽  
Promoter Activity ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Minimal Promoter ◽  
Intestinal Cell ◽  
Epithelial Cell Line ◽  
Hek 293

The mechanism of biotin uptake in human intestine has been well characterized and involves the human sodium-dependent multivitamin transporter (hSMVT), yet little is known about the molecular/transcriptional regulation of the system. Previous investigations cloned the 5′ regulatory region of the hSMVT gene and identified the minimal promoter. To expand these investigations, we compared activity of the hSMVT promoter in three human intestinal epithelial cell lines (NCM460, Caco-2, and HuTu-80) and contrasted a renal epithelial cell line (HEK-293). We analyzed the role of putative cis-elements in regulating promoter activity and confirmed activity of the cloned hSMVT promoter in vivo. In vitro studies demonstrated that all cell lines utilized the same minimal promoter region, and mutation of specific cis-regulatory elements [Kruppel-like factor 4 (KLF-4) and activator protein-2 (AP-2)] led to a decrease in promoter activity in all intestinal cell types but not in renal cells. Using electrophoretic mobility shift assays, we identified two specific DNA/protein complexes. Using oligonucleotide competition and antibody supershift analysis, we determined that KLF-4 and AP-2 were involved in forming the complexes. In HEK-293 cells, overexpressing KLF-4 increased the endogenous hSMVT message levels threefold and activated a cotransfected hSMVT promoter-reporter construct. In vivo studies using hSMVT promoter-luciferase transgenic mice established physiological relevance and showed the pattern of hSMVT promoter expression to be similar to endogenous mouse SMVT mRNA expression. The results demonstrate, for the first time, the importance of KLF-4 and AP-2 in regulating the activity of the hSMVT promoter in the intestine and provide direct in vivo confirmation of hSMVT promoter activity.

A villous cell-derived inhibitor of intestinal cell proliferation

1981 ◽  
Vol 241 (6) ◽  
pp. G520-G527
Author(s):  
R. J. May ◽  
A. Quaroni ◽  
K. Kirsch ◽  
K. J. Isselbacher
Keyword(s):  
Epithelial Cell ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Intestinal Epithelial Cell ◽  
Amino Acid Uptake ◽  
Intestinal Cell ◽  
Epithelial Cell Line ◽  
Acid Uptake ◽  
Intestinal Epithelial

To test the hypothesis that the intestinal villous cell synthesizes a mitotic inhibitor that is specific for crypt cells, we have partially purified an extract from rat intestinal villous cells (VCE) and have demonstrated that it strongly and reversibly inhibits cell division and DNA synthesis in an intestinal epithelial cell line (IEC-6 cells). VCE produced a 60--70% inhibition of [3H]thymidine incorporation into DNA and a similar magnitude of inhibition of labeling of nuclei in autoradiographic studies. This inhibition was not associated with cytotoxicity as assessed by the effect of VCE on 51Cr release, hexose or amino acid uptake, and protein synthesis. VCE appears specific for IEC-6 cells because it did not affect DNA synthesis in 10 other cell lines, and extracts derived from other cell lines and from colonic mucosa did not affect DNA synthesis in IEC-6 cells. VCE may represent a villous cell factor involved in the control of intestinal epithelial cell turnover in vivo.

Characterization of the 5′-regulatory region of the human thiamin transporter SLC19A3: in vitro and in vivo studies

2004 ◽  
Vol 287 (4) ◽  
pp. G822-G829 ◽  
Author(s):  
Svetlana M. Nabokina ◽  
Hamid M. Said
Keyword(s):  
Promoter Region ◽  
Promoter Activity ◽  
Critical Role ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Minimal Promoter ◽  
Minimal Promoter Region

Transcriptional regulation of expression of the human thiamin transporter-2 (the product of the SLC19A3 gene) is unknown. In this study, we cloned the 5′-regulatory region of the human SLC19A3 gene (2,016 bp), identified the minimal promoter region required for basal activity, demonstrated a critical role for specific cis-regulatory elements in determining the promoter activity, and confirmed activity and physiological relevance of the cloned SLC19A3 promoter in vivo. With the use of transiently transfected human intestinal epithelial Caco-2 cells and 5′-deletion analysis, the minimal promoter region required for basal activity of the SLC19A3 promoter was found to be encoded in a sequence between −77 and +59 by using the start of transcription initiation as position 1. This minimal region was found to contain a number of putative cis-regulatory elements, with a critical role for a stimulating protein-1 (SP1)/GC-box binding site (at position −48/−45 bp) established by means of mutational analysis. With the use of EMSA and supershift assays, the binding of SP1 and SP3 to the minimal promoter region was also demonstrated. In transiently transfected Drosophila SL2 cells, both SP1 and SP3 transactivated the SLC19A3 minimal promoter in a dose-dependent manner and in combination demonstrated an additive stimulatory effect. Functionality of the full-length SLC19A3 promoter was confirmed in vivo in transgenic mice expressing the promoter-luciferase reporter gene. These studies report the first characterization of the SLC19A3 promoter in vitro and in vivo and demonstrate the importance of an SP1 cis-regulatory element in regulating promoter activity of this important human gene.

scholarly journals Identification and analysis of the promoter region of the human methionine sulphoxide reductase A gene

2005 ◽  
Vol 393 (1) ◽  
pp. 321-329 ◽  
Author(s):  
Antonella De Luca ◽  
Paolo Sacchetta ◽  
Carmine Di Ilio ◽  
Bartolo Favaloro
Keyword(s):  
Cell Lines ◽  
Transcription Initiation ◽  
Promoter Region ◽  
Promoter Activity ◽  
Regulatory Region ◽  
Initiation Site ◽  
Luciferase Reporter ◽  
Mcf7 Cells ◽  
Luciferase Reporter Gene ◽  
Hek 293

MsrA (methionine sulphoxide reductase A) is an antioxidant repair enzyme that reduces oxidized methionine to methionine. Moreover, the oxidation of methionine residues in proteins is considered to be an important consequence of oxidative damage to cells. To understand mechanisms of human msrA gene expression and regulation, we cloned and characterized the 5′ promoter region of the human msrA gene. Using 5′-RACE (rapid amplification of cDNA ends) analysis of purified mRNA from human cells, we located the transcription initiation site 59 nt upstream of the reference MsrA mRNA sequence, GenBank® accession number BC 054033. The 1.3 kb of sequence located upstream of the first exon of msrA gene was placed upstream of the luciferase reporter gene in a pGL3-Basic vector and transfected into different cell lines. Sequentially smaller fragments of the msrA promoter region were generated by PCR, and expression levels were monitored from these constructs within HEK-293 and MCF7 human cell lines. Analysis of deletion constructs revealed differences in promoter activity in these cell lines. In HEK-293 cells, the promoter activity was constant from the minimal promoter region to the longest fragment obtained. On the other hand, in MCF7 cells we detected a down-regulation in the longest fragment. Mutation of a putative negative regulatory region that is located between −209 and −212 bp (the CCAA box) restored promoter activity in MCF7 cells. The location of the msrA promoter will facilitate analysis of the transcriptional regulation of this gene in a variety of pathological contexts.

In vitro and in vivo characterization of the minimal promoter region of the human thiamin transporter SLC19A2

2003 ◽  
Vol 285 (3) ◽  
pp. C633-C641 ◽  
Author(s):  
Jack C. Reidling ◽  
Hamid M. Said
Keyword(s):  
Transgenic Mice ◽  
Mammalian Cells ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Regulatory Region ◽  
Minimal Promoter ◽  
Human Tissues ◽  
Cis Elements

The molecular mechanisms involved in the regulation of thiamin transport in mammalian cells are poorly understood. Previous studies established that a human thiamin transporter, SLC19A2, plays a role in thiamin uptake in human tissues. We cloned the 5′ regulatory region of the SLC19A2 gene, identified the minimal promoter required for basal activity, and located multiple putative cis elements. To further characterize the SLC19A2 promoter, we investigated, in the present study, the role of the putative cis elements in regulating the activity of the SLC19A2 promoter in vitro and confirmed the activity of the SLC19A2 promoter in vivo. In vitro studies demonstrated that mutation of specific cis elements in the SLC19A2 minimal promoter [Gut-enriched Krupple-like factor (GKLF), nuclear factor-1 (NF-1), and stimulating protein-1 (SP-1)] led to a decrease in activity. Using electrophoretic mobility shift assays, four specific DNA/protein complexes were identified. The interacting factors were determined by oligonucleotide competition and antibody supershift analysis and shown to be GKLF, NF-1, and SP-1. Cotransfection studies of the SLC19A2 promoter with an SP-1 containing vector in Drosophila SL2 cells further confirmed a role for SP-1 in regulating SLC19A2 promoter activity. In vivo studies using transgenic mice established the functionality of the full-length and minimal SLC19A2 promoters. Furthermore, our studies revealed that the pattern of expression of the SLC19A2 promoter-Luciferase constructs in transgenic mice was similar to the reported SLC19A2 RNA expression pattern in native human tissues. The results demonstrate the importance of GKLF, NF-1, and SP-1 in regulating the activity of the SLC19A2 promoter and provide direct in vivo confirmation of promoter activity.

scholarly journals Identification and characterization of the minimal 5′-regulatory region of the human riboflavin transporter-3 (SLC52A3) in intestinal epithelial cells

2015 ◽  
Vol 308 (2) ◽  
pp. C189-C196 ◽  
Author(s):  
Abhisek Ghosal ◽  
Subrata Sabui ◽  
Hamid M. Said
Keyword(s):  
Epithelial Cells ◽  
Transgenic Mice ◽  
Promoter Activity ◽  
Intestinal Epithelial Cells ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Intestinal Epithelial ◽  
Identification And Characterization

The human riboflavin (RF) transporter-3 (product of the SLC52A3 gene) plays an important role in intestinal RF absorption. Our aims in this study were to identify the minimal 5′-regulatory region of the SLC52A3 gene and the regulatory element(s) involved in its activity in intestinal epithelial cells, as well as to confirm promoter activity and establish physiological relevance in vivo in transgenic mice. With the use of transiently transfected human intestinal epithelial HuTu 80 cells and 5′-deletion analysis, the minimal SLC52A3 promoter was found to be encoded between −199 and +8 bp (using the start of the transcription start site as position 1). Although several putative cis-regulatory elements were predicted in this region, only the stimulating protein-1 (Sp1) binding site (at position −74/−71 bp) was found to play a role in promoter activity, as indicated by mutational analysis. Binding of Sp1 to the minimal SLC52A3 promoter was demonstrated by means of EMSA and supershift assays and by chromatin immunoprecipitation analysis. Studies with Drosophila SL2 cells (which lack Sp activity) confirmed the importance of Sp1 in driving the activity of the SLC52A3 minimal promoter; they further showed that Sp3 can also do the activation. Finally, with the use of luciferase gene fusions, the activity of the cloned SLC52A3 promoter was confirmed in vivo in transgenic mice. These studies report, for the first time, on the identification and characterization of the SLC52A3 promoter and also demonstrate the importance of Sp1 in regulating its activity in intestinal epithelial cells.

scholarly journals Goblet-cell-specific transcription of mouse intestinal trefoil factor gene results from collaboration of complex series of positive and negative regulatory elements

1999 ◽  
Vol 341 (2) ◽  
pp. 461-472 ◽  
Author(s):  
Hiroshi ITOH ◽  
Nagamu INOUE ◽  
Daniel K. PODOLSKY
Keyword(s):  
Cell Lines ◽  
Goblet Cell ◽  
Regulatory Element ◽  
Regulatory Region ◽  
Goblet Cells ◽  
Regulatory Elements ◽  
Intestinal Cell ◽  
Gene Promoters ◽  
Trefoil Factor ◽  
Intestinal Trefoil Factor

Intestinal trefoil factor (ITF) is expressed selectively in intestinal goblet cells. Previous studies of the rat ITF gene identified one cis-regulatory element, designated the goblet-cell-response element (GCRE), present in the proximal region of the promoter. To identify additional cis-regulatory elements responsible for goblet-cell-specific expression, a DNA fragment containing 6353 bp of the 5′-flanking region of the mouse ITF gene was cloned and its promoter activity was examined extensively. In human and murine intestinal-derived cell lines (LS174T and CMT-93), the luciferase activities of a 6.3-kb construct were 5- and 2-fold greater than the smaller 1.8-kb construct, respectively. In contrast, the activity in non-intestinal cell lines (HepG2 and HeLa) was 2-4-fold lower than the smaller construct. In the region downstream from the 1.8-kb position, strong luciferase activities in LS174T and HepG2 cells were observed using a 201-bp construct. Interestingly, increased activity was almost completely suppressed in cells transfected with a 391-bp construct. Detailed analyses of this region revealed the existence of a 11-bp positive regulatory element (-181 to -170; ACCTCTTCCTG) and a 9-bp negative regulatory element (-208 to -200; ATTGACAGA) in addition to the GCRE. All three elements were well conserved among human, rat and mouse ITF gene promoters. In addition, a mutant 1.8-kb construct in which the negative regulatory region was deleted yielded the same approximate luciferase activity as a 6.3-kb construct, suggesting binding of a goblet-cell-specific silencer inhibitor (SI) between -6.3 and -1.8 kb. The SI present in goblet cells may block the silencers' binding to the pre-initiation complex and allow increased transcriptional activity driven by specific and non-specific enhancers. High-level expression of the mouse ITF gene specifically in intestinal goblet cells may be achieved through the combined effects of these regulatory elements.

Molecular mechanisms of epithelial cell-specific expression and regulation of the human anion exchanger (pendrin) gene

2008 ◽  
Vol 294 (5) ◽  
pp. C1261-C1276 ◽  
Author(s):  
Lior Adler ◽  
Edna Efrati ◽  
Israel Zelikovic
Keyword(s):  
Epithelial Cell ◽  
Inner Ear ◽  
Cell Lines ◽  
Anion Exchanger ◽  
Promoter Activity ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Acidic Ph ◽  
Alkaline Ph ◽  
Specific Expression

Pendrin, a Cl−/anion exchanger encoded by the gene PDS, is highly expressed in the kidney, thyroid, and inner ear epithelia and is essential for bicarbonate secretion, iodide accumulation, and endolymph ion balance, respectively. This study aimed to define promoter regulatory elements essential for renal, thyroid, and inner ear epithelial cell-specific expression of human PDS (hPDS) and to explore the effect of ambient pH and aldosterone on hPDS promoter activity. Endogenous pendrin mRNA and protein were detected in renal HEK293, thyroid LA2, and inner ear VOT36 epithelial cell lines, but not in the fibroblast cell line, NIH3T3. A 4.2-kb hPDS 5′-flanking DNA sequence and consecutive 5′-deletion products were cloned into luciferase reporter vectors and transiently transfected into the above cell lines. Distinct differences in expression/activity of deduced positive/negative regulatory elements within the hPDS promoter between HEK293, LA2, and VOT36 cells were demonstrated, with only basal activity in NIH3T3 cells. Acidic pH (7.0–7.1) decreased and alkaline pH (7.6–7.7) increased hPDS promoter activity in transfected HEK293 and VOT36, but not in LA2 cells. Aldosterone (10−8 M) reduced hPDS promoter activity in HEK293 but had no effect in LA2 and VOT36 cells. These pH and aldosterone-induced effects on the hPDS promoter occurred within 96-bp and 89-bp regions, respectively, which likely contain distinct response elements to these modulators. Acidic pH and aldosterone decreased, and alkaline pH increased, endogenous pendrin mRNA level in HEK293 cells. In conclusion, pendrin-mediated HCO3− secretion in the renal tubule and anion transport in the endolymph may be regulated transcriptionally by systemic pH and aldosterone.

Electrophysiological Measurements of a Toad Renal Epithelial Cell Line (A6) as an Assay for Predicting Ocular Eye Irritancy

1992 ◽  
Vol 20 (2) ◽  
pp. 218-221
Author(s):  
Henning F. Bjerregaard
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
South African ◽  
Mdck Cells ◽  
Epithelial Cell Line ◽  
Renal Epithelial Cell ◽  
Electrophysiological Measurements ◽  
Renal Epithelial Cell Line ◽  
Darby Canine Kidney

An established epithelial cell line (A6) from a South African clawed toad (Xenopus laevis) kidney was used as a model for the corneal epithelium of the eye in order to determine ocular irritancy. When grown on Millipore filter inserts, A6 cells form a monolayer epithelium of high electrical resistance and generate a trans-epithelial potential difference. These two easily-measured electrophysiological endpoints showed a dose-related decrease after exposure for 24 hours to seven selected chemicals of different ocular irritancy potential. It was demonstrated that both trans-epithelial resistance and potential ranked closely with in vivo eye irritancy data and correlated well (r = 0.96) with loss of trans-epithelial impermeability of Madin-Darby canine kidney (MDCK) cells, detected by use of a fluorescein leakage assay.

scholarly journals Probing the Intracellular Bio-Nano Interface in Different Cell Lines with Gold Nanostars

Nanomaterials ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1183
Author(s):  
Cecilia Spedalieri ◽  
Gergo Péter Szekeres ◽  
Stephan Werner ◽  
Peter Guttmann ◽  
Janina Kneipp
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Early Stage ◽  
Protein Corona ◽  
Fibroblast Cell ◽  
Ultrastructural Level ◽  
Epithelial Cell Line ◽  
Animal Cells ◽  
Gold Nanostars

Gold nanostars are a versatile plasmonic nanomaterial with many applications in bioanalysis. Their interactions with animal cells of three different cell lines are studied here at the molecular and ultrastructural level at an early stage of endolysosomal processing. Using the gold nanostars themselves as substrate for surface-enhanced Raman scattering, their protein corona and the molecules in the endolysosomal environment were characterized. Localization, morphology, and size of the nanostar aggregates in the endolysosomal compartment of the cells were probed by cryo soft-X-ray nanotomography. The processing of the nanostars by macrophages of cell line J774 differed greatly from that in the fibroblast cell line 3T3 and in the epithelial cell line HCT-116, and the structure and composition of the biomolecular corona was found to resemble that of spherical gold nanoparticles in the same cells. Data obtained with gold nanostars of varied morphology indicate that the biomolecular interactions at the surface in vivo are influenced by the spike length, with increased interaction with hydrophobic groups of proteins and lipids for longer spike lengths, and independent of the cell line. The results will support optimized nanostar synthesis and delivery for sensing, imaging, and theranostics.

scholarly journals CircTP63 promotes hepatocellular carcinoma progression by sponging miR-155-5p and upregulating ZBTB18

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jiantao Wang ◽  
Jinbiao Che
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Lines ◽  
Circular Rnas ◽  
Epithelial Cell Line ◽  
High Morbidity ◽  
Mrna Levels ◽  
Promoting Effect ◽  
Tumor Stages ◽  
Hcc Cells

Abstract Background Hepatocellular carcinoma (HCC) is the leading cause of tumor-related death worldwide due to high morbidity and mortality, yet lacking effective biomarkers and therapies. Circular RNAs (circRNAs) are a group of non-coding RNAs that regulate gene expression through interacting with miRNAs, implicating in the tumorigenesis and progression. A novel circRNA, circTP63, was reported to be an oncogene in HCC. However, its role in HCC remains unclear. Methods qRT-PCR was used to assess the mRNA levels of CircTP63 in 90 pairs of tumor and adjacent normal tissues from HCC patients, one human normal hepatic epithelial cell line and HCC cell lines. CCK-8, colony formation, transwell, and flow cytometry assays were performed to detect the cellular function of circTP63/miR-155-5p/ZBTB18 in HCC cells. HCC xenograft mice models were established to assess the in vivo effect of circTP63. Bioinformatic analysis, RNA pull-down and luciferase assays were used to determine the interaction among circTP63/miR-155-5p/ZBTB18. Results circTP63 was significantly upregulated in HCC tissues and cell lines. High circTP63 expression is closely associated with the tumor stages, lymph node metastasis, and poor prognosis of HCC patients. Functionally, knockdown of circTP63 inhibited cell proliferation, migration, invasion, and promoted cell apoptosis of HCC. Meanwhile, overexpression of circTP63 enhanced HCC progression. Mechanically, circTP63 was a sponge of miR-155-5p to facilitate the ZBTB18 expression, and the ZBTB18 expression in HCC tissues was negatively associated with the survival rate of HCC patients. Furthermore, rescued assays revealed that the reduced tumor-promoting effect on HCC cells induced by knockdown of circTP63 can be reversed by miR-155-5p inhibitor or ZBTB18 overexpression. Conclusion Our data highlight a critical circTP63-miR-155-5p-ZBTB18 regulatory network involved in the HCC progression, gaining mechanistic insights into the function of circRNAs in HCC progression, and providing effective biomarkers and therapeutic targets for HCC treatment.

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