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T S Elton

Publications and source records attributed to T S Elton.

At least 19 recordsLinked to original sources
PubMed2001

The transcription factors Sp1 and Sp3 are required for human angiotensin II type 1 receptor gene expression in H295-R cells.

The peptide hormone angiotensin II regulates a variety of physiological responses which are mediated by its interaction with high affinity G protein-coupled receptors localized on the surface of target cells. Our previous studies have demonstrated that a 145 bp sequence within the promoter region was required for basal level expression of the human angiotensin II type 1 receptor (hAT(1)R) gene. In the present study, deletional analysis of the hAT(1)R promoter localized the major regulatory sequence to two overlapping GC boxes harbored within the -105 to -85 bp region relative to the transcription start site in H295-R cells. Electrophoretic mobility shift assays (EMSAs) using a double-stranded (ds) oligonucleotide corresponding to this region and H295-R cell nuclear extract resulted in five specific DNA-protein complexes. EMSAs performed with competitive ds-oligonucleotides which harbored the consensus binding site for Sp1 prevented the formation of the DNA-protein complexes. Supershift EMSAs also demonstrated that Sp1 and Sp3 could bind to the GC boxes present within the -105 to -85 bp region of the hAT(1)R promoter. Transactivation experiments utilizing Drosophila SL2 cells, which lack endogenous Sp family transcription factors, demonstrated that Sp1 and Sp3 activated the hAT(1)R promoter and that maximal activation was only achieved when both GC boxes were present. Taken together, these findings suggest that Sp1 and Sp3 are necessary for the expression of the hAT(1)R gene in H295-R cells.

Acetaldehyde↗
PubMed2001

Identification and characterization of functional angiotensin II type 1 receptors on immortalized human fetal aortic vascular smooth muscle cells.

Studies investigating the mechanisms that govern the expression of the human angiotensin II type 1 receptor (hAT(1)R) gene have progressed slowly due to the lack of human cell lines that express the AT(1)R. Recently, however, an immortalized human fetal aortic vascular smooth muscle cell line (FLTR) was generated using an amphotropic recombinant retroviral construct containing the E6/E7 open reading frames of the human papillomavirus type 16. Radioligand binding studies were undertaken to determine whether angiotensin II (Ang II) receptors were expressed on these cells. FLTR cell membranes were shown to express high-affinity Ang II receptors having a B(max) value of 324+/-43 fmol/mg protein and a K(d) of 0.36+/-0.1 nM. In both membranes and intact cells, Ang II, Ang III and the selective AT(1)R antagonist, Losartan, all had a high affinity for the receptor, suggesting that FLTR cells express the AT(1)R subtype. The expression of the hAT(1)R was validated by Northern and Western blot and RT-PCR experiments. In intact FLTR cells, Ang II (100 nM) evoked an increase in intracellular calcium ([Ca(2+)](i)) and induced hyperplasia. Additionally, our results demonstrated that FLTR cells were readily transfected, and hAT(1)R promoter luciferase constructs exhibited robust promoter activity (i.e. approximately 22-fold increase over pGL3-Basic only). Finally, our results demonstrated that the hAT(1)R gene is differentially regulated in FLTR cells vs. H295-R cells, a human adrenocarcinoma cell line that also abundantly expresses the AT(1)R. Taken together, our results suggest that FLTR cells express functional AT(1)Rs and will provide an excellent model system in which to investigate hAT(1)R gene regulation.

Angiotensins↗
PubMed2001

Human angiotensin II type 1 receptor isoforms encoded by messenger RNA splice variants are functionally distinct.

Human tissues that express the angiotensin II (Ang II) type 1 receptor (hAT(1)R) can synthesize four distinct alternatively spliced hAT(1)R mRNA transcripts. In this study, we show that the relative abundance of these mRNA transcripts varies widely in human tissues, suggesting that each splice variant is functionally distinct. Here we demonstrate, for the first time, that the hAT(1)R-B mRNA splice variant encodes a novel long hAT(1)R isoform in vivo that has significantly diminished affinity for Ang II (i.e. >3-fold) when compared with the short hAT(1)R isoform (encoded by hAT(1)R-A mRNA splice variant). This reduced agonist affinity caused a significant shift to the right in the dose-response curve for Ang II-induced inositol trisphosphate production and Ca(2+) mobilization of the long hAT(1)R when compared with that of the short hAT(1)R. The functional differences between these isoforms allows Ang II responsiveness to be fine-tuned by regulating the relative abundance of the long and short hAT(1)R isoform expressed in a given human tissue.

Adrenal Cortex Neoplasms↗
PubMed2000

Basal level transcriptional regulation of the human angiotensin II type 1 receptor gene.

The peptide hormone angiotensin II regulates a variety of physiological responses which are mediated by its interaction with high affinity G protein-coupled receptors localized on the surface of target cells. To gain insights into the transcriptional regulation of the human angiotensin II type 1 receptor (hAT(1)R) gene, we have isolated 1 kb of the 5'-flanking sequence of this gene. Expression constructs containing various 5'-deletions of the hAT(1)R promoter region, fused upstream to the luciferase reporter gene, were transiently transfected into H295-R, HEC-1B and A549 cells. It was demonstrated that a 145 bp sequence within the promoter region was required for basal level expression of the hAT(1)R gene in all of the three cell lines investigated. Computer analysis indicated the existence of numerous putative transcription factor binding sites in this region. Further detailed deletion data suggested essential transcription factor binding sites between -98 and -79 bp. Electrophoretic mobility shift assays revealed that four protein-DNA complexes were formed within the -98 to -79 bp region of the hAT(1)R gene when incubated with H295-R cell nuclear extract. Site-directed mutagenesis experiments showed that a putative Sp1 binding site was critical for the basal level expression of the hAT(1)R gene.

Base Sequence↗
PubMed2000

Molecular cloning and characterization of the human phosducin-like protein (hPhLP) promoter.

Phosducin-like protein (PhLP) is an inducible Gbetagamma binding protein which is hypothesized to be a ubiquitous G protein regulator. To elucidate the mechanisms regulating the expression of the human PhLP (hPhLP) gene, we have cloned and characterized its 5'-flanking region. Primer extension analysis identified a major transcription initiation site 172 bp upstream of the ATG start codon. Analysis of the 5'-flanking region revealed that, although it lacked a TATA box element, the hPhLP promoter did contain several consensus binding motifs including AP4, CCAAT, CREB, NF-kappaB, SP1 and E2F. Transient transfection analyses using a series of 5'-flanking deletion/luciferase reporter gene constructs identified a 25 bp sequence (-80 to -55 bp) that is necessary for basal level transcription of the hPhLP gene in all the cell lines investigated. Interestingly, dependent upon the cell line, distinct transcription factors bind to this region suggesting that basal level hPhLP gene transcription may be regulated in a tissue-specific manner.

Base Sequence↗
PubMed2000

Characterization of the promoter-directing expression of growth hormone in a monocyte cell line.

Previous work from our laboratory has shown that cells of the immune system produce a growth hormone (GH) molecule similar to that produced by the pituitary. In the present study, using Southern analysis of RT-PCR products and sequencing of cloned cDNA molecules, we demonstrate that lymphoid cell lines utilize the same promoter and first exon as the pituitary somatotrope. To identify the cis-elements involved in transcriptional regulation of immune cell-derived GH, we have coupled rat GH promoter fragments to a luciferase reporter gene and transfected a monocyte cell line (P-388) by electroporation. The results suggest the presence of both positive (-299/-193 bp) and negative (-193/-107 bp) regulatory elements. The same constructs transfected in the pituitary cell line, GH3, in contrast to the monocyte cell line, showed a gradual decrease in luciferase expression. The overexpression of GHF-1 or GHF-2 resulted in a modest but significant reduction in rat GH promoter activity in the P-388 cell line. Taken together, the data suggest that immune cells utilize the same first exon and promoter sequence for the expression of monocyte GH as that reported for the expression of pituitary GH. Further, it appears that sequences between -299 and -107 bp are important in the regulation of the promoter where different transcription factors may be recruited to promote GH expression in a monocyte cell line.

Animals↗
PubMed1999

Human phosducin-like protein (hPhLP) messenger RNA stability is regulated by cis-acting instability elements present in the 3'-untranslated region.

Phosducin (Pd) and phosducin-like protein (PhLP) have been shown to regulate G-protein signaling by binding G beta gamma subunits. To better define the function and regulation of PhLP, and to begin to investigate its potential role in human pathophysiological states, we have cloned the human PhLP (hPhLP) cDNA. The hPhLP shows 92% identity with the rat PhLP (rPhLP). However, unlike the rPhLP, no evidence of hPhLP isoforms were detected in the human tissues investigated. Additionally, unlike the rPhLP, alternative polyadenylation sites were detected in hPhLP cDNA clones which corresponded with two distinct mRNA transcripts, 1.2 kb and 3.1 kb, respectively. Interestingly, the predominantly expressed long transcript contains multiple AU-rich elements (AREs) in its 3'-untranslated region (3'-UTR) which have been shown to correlate with rapid mRNA turnover and translational control. This study shows that the hPhLP AREs are functional both in vitro and in vivo, with the long transcript exhibiting a much shorter mRNA half-life. We also demonstrate that subcloning of either the full-length 3'-UTR or the ARE-rich region of the long transcript immediately following the stop codon of luciferase reporter gene confers instability to the luciferase mRNA and results in a ninefold reduction of luciferase activity in the cell types investigated. Taken together, these findings suggest that the AREs present in the long hPhLP mRNA may play a critical role in the regulation of hPhLP gene expression.

Amino Acid Sequence↗
PubMed1999

An A/T-rich cis-element is essential for rat angiotensin II type 1A receptor transcription in vascular smooth muscle cells.

Transcriptional mechanisms regulating the expression of the rat angiotensin II type 1A receptor (rAT1AR) gene were investigated in cultured rat vascular smooth muscle cells (VSMC). Transcriptional analyses of various 5'-deletion mutants of the rAT1AR promoter region, fused upstream from the firefly luciferase gene, demonstrated that a 71 base pair (bp) region (-557 to -486 bp, with respect to transcription initiation) was necessary for expression of this gene in VSMC. Electrophoretic mobility shift assays demonstrated that specific protein-DNA complexes were formed with the -516 to -486 bp region of the rAT1AR promoter when incubated with VSMC extract. Computer analysis of this region indicated the presence of an A/T-rich sequence (i.e., TTTAAAAATAAA) which is similar to a myocyte enhancer binding factor 2 (MEF2) cis-regulatory element (i.e., CTTAAAAATAAC). Site-directed mutagenesis of this A/T-rich sequence inhibited rAT1AR promoter activity in VSMC, suggesting that this region was necessary for expression of this gene in these cells. Immuno-gel shift experiments suggest that MEF2 heterodimers may interact with the A/T-rich sequence in the rAT1AR promoter. Additionally, it was demonstrated that a transcription factor non-homologous to MEF2 can also interact with this A/T-rich site in the rAT1AR promoter. Taken together, our results suggest that MEF2 heterodimers, and/or transcription factors non-homologous to MEF2, are required to regulate the expression of the rAT1AR gene in VSMC.

Animals↗
PubMed1995

A functional comparison of the rat type-1 angiotensin II receptors (AT1AR and AT1BR).

To evaluate and functionally compare the rat AT1A and AT1B receptor subtypes, stable Chinese hamster ovary (CHO) cell lines expressing either recombinant receptor in approximately equal numbers were generated. Radioligand binding data suggests that the recombinant AT1A receptor is pharmacologically similar to the recombinant AT1B receptor. Functional studies indicate that both receptor subtypes can independently activate the phospholipase C/IP3 and the dihydropyridine-sensitive voltage-dependent Ca2+ channel signal transduction pathways with equal efficiency, but are unable to modulate cAMP accumulation under our experimental conditions. Furthermore, both receptors can be directly involved in the cellular growth properties of AII. Slot-blot experiments clearly demonstrate that these receptors are expressed in a tissue-specific manner. A sequence comparison of the 5' flanking regions of these two genes shows that they have very little sequence homology (approximately 36%), suggesting that although the AT1A and AT1B receptors appear to be pharmacologically and functionally similar, the control of their expression seems to be governed by distinct transcription factors.

Animals↗
PubMed1995

Molecular cloning of a cDNA encoding putative molt-inhibiting hormone from the blue crab, Callinectes sapidus.

A cDNA library was constructed using poly(A+) RNA isolated from eyestalk neural ganglia of the blue crab, Callinectes sapidus. The library was screened using a probe generated by PCR based on the published amino acid sequence of molt-inhibiting hormone from the shore crab, Carcinus maenas. DNA sequence analysis of one positive clone revealed a 339 bp open reading frame encoding a 78-residue putative molt-inhibiting hormone and a 35-residue signal peptide. The deduced amino acid sequence of C. sapidus molt-inhibiting hormone is 79% homologous with that of C. maenas. Northern blot analysis, using a fragment of the cloned molt-inhibiting hormone cDNA as probe, revealed specific hybridization to a single band (approximately 1.4 kb) in RNA extracted from eyestalk but not control tissues.

Amino Acid Sequence↗
PubMed1995

The sequence and genomic organization of the human type 2 angiotensin II receptor.

A human genomic DNA library was screened utilizing a human angiotensin II type 2 receptor (hAT2R) cDNA as a probe. Several positive clones were isolated and characterized. A comparison of the hAT2R cDNA sequence with the hAT2R genomic clone sequence suggests that the hAT2R gene is composed of three exons and spans at least 5 kb. Exons 1 and 2 encode for 5' untranslated mRNA sequence and exon 3 harbors the entire uninterrupted open reading frame of the hAT2R. Sequence analysis of the 5'-flanking region of the hAT2R gene demonstrates that it contains the typical sequence motifs found in many eukaryotic promoters. Interestingly, however, this promoter region also includes an interferon consensus sequence binding protein site (ICSBP) and a putative embryonal, long terminal repeat binding protein (ELP) site. The presence of these novel putative transcription factor binding sites suggests that this gene may be regulated in a unique manner.

Animals↗
PubMed1995

Hyperglycemia alters cytoplasmic Ca2+ responses to capacitative Ca2+ influx in rat aortic smooth muscle cells.

Concentrations of free cytoplasmic Ca2+ in rat aortic smooth muscle (RASM) cells were monitored using the ratiometric Ca2+ indicator fura 2-acetoxymethyl ester (AM). In RASM cells cultured in 5 mM Glc, incubation with angiotensin II, ATP, or thapsigargin [a selective inhibitor of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase] depleted SR Ca2+ stores and initiated a capacitative Ca2+ influx through the plasma membrane. This influx was resistant to verapamil, a selective inhibitor of L-type voltage-gated Ca2+ channels, but was sensitive to SKF-96365, an inhibitor of the receptor-operated Ca2+ entry pathway. RASM cells cultured in 25 mM Glc exhibited a significant decrease in cytoplasmic Ca2+ responses to agonist-induced Ca2+ release from SR stores and to subsequent capacitative Ca2+ entry. In addition, the cytoplasmic response to thapsigargin-induced release of Ca2+ from the SR in hyperglycemic cells peaked more sharply than in control cells and returned to baseline more rapidly. The effects of hyperglycemia were not overcome by myo-inositol supplementation.

Adenosine Triphosphate↗
PubMed1995

Endothelin-A receptor antagonist prevents acute hypoxia-induced pulmonary hypertension in the rat.

Exposure to hypoxia is associated with increased pulmonary artery pressure and plasma endothelin-1 (ET-1) levels and with selective enhancement in ET-1 peptide and mRNA and endothelin-A (ETA) receptor mRNA levels in rat lung. The current study tested the hypothesis that endogenous ET-1 can account for hypoxia-induced pulmonary hypertension via a paracrine effect on ETA receptors in lung. Intravenous infusion of the ETA receptor antagonist BQ-123 (D-Trp-D-Asp-Pro-D-Val-Leu) (0.4 mg/microliters at 1 microliter/h) into Sprague-Dawley rats beginning 4 h before and for 90 min during normobaric hypoxia (10% O2) markedly attenuated the hypoxic response: mean pulmonary artery pressure increased from 17.2 +/- 0.7 to 29.0 +/- 1.2 mmHg in saline control rats but did not increase from baseline in BQ-123-treated rats. BQ-123 did not alter systemic arterial pressure, heart rate, or plasma endothelin-1 levels. These findings suggest that ET-1 synthesized in lung in response to hypoxia acts locally on ETA receptors to cause pulmonary hypertension.

Acute Disease↗
PubMed1995

Selective downregulation of ANP-clearance-receptor gene expression in lung of rats adapted to hypoxia.

To test the hypothesis that expression of atrial natriuretic peptide (ANP)-receptor genes is modified to provide a compensatory mechanism against hypoxic pulmonary hypertension, steady state mRNA levels for the ANP-A receptor (or guanylate cyclase-A; ANPAR), ANP-B receptor (or guanylate cyclase-B; ANPBR), and ANP-clearance receptor (ANPCR) were quantitated by Northern blot and slot-blot analysis in lung, kidney, spleen, and liver of hypoxia-adapted rats and air controls. Exposure of rats to short-term (48 h) and chronic (4 wk) hypoxia (10% O2, 1 atm) did not affect lung ANPAR-mRNA levels. Lung ANPBR-mRNA levels were unchanged by short-term hypoxia but selectively increased (approximately twofold) by chronic hypoxia. ANPCR-mRNA levels were selectively and significantly downregulated by 48-h and 4-wk hypoxia in lung but were unchanged or upregulated in other tissues. Lung ANPCR gene transcription, assessed by nuclear-runoff analysis, was decreased by hypoxia. These data support the conclusion that altered pulmonary ANP-receptor gene expression modulates the development of hypoxic pulmonary hypertension.

Adaptation, Physiological↗
PubMed1995

Calcium-sensitive chloride channels in vascular smooth muscle cells.

Chloride (Cl-) channels were characterized in vascular smooth muscle cells (VSMC) using radioisotope flux and patch-clamp electrophysiological techniques. Transmembrane 125iodine (125I) efflux from subcultured (Passage 1-5) rat aortic VSMCs was used as an indicator of Cl- movements to study the relationship between intracellular calcium concentration ([Ca2+]i) and Cl- channel activity. Angiotensin II (Ang II) (10(-7) M) and adenosine 5'-triphosphate (ATP) (10(-4) M) induced rapid increases (9.7- and 14.9-fold, respectively) in 125I efflux rates. We found that both Ang II- and ATP-stimulated 125I efflux and [Ca2+]i increases were completely abolished after brief incubation (20 microM, 20 min) with the acetoxymethyl ester of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), a membrane-permeable Ca2+ chelator. However, when external EGTA was used to blunt agonist-stimulated Ca2+ influx, 125I efflux was still increased in response to Ang II and ATP. These data suggest that Ca2+ release from intracellular sites is sufficient to activate Cl- channels in response to Ang II and ATP. Using standard patch-clamp electrophysiological techniques, we found that Ang II, a Ca(2+)-mobilizing agonist, stimulated outward Cl- currents (gCl = 75 pS) in cell-attached (C/A) patches of primary and subcultured VSMCs. Collectively, these data suggest that Ang II and other vasoconstrictor agents stimulate Cl- channel activity via increases in [Ca2+]i. Cl- channel activation may help to depolarize the VSMC membrane leading to increased Ca2+ influx during agonist stimulation.

Animals↗
PubMed1994

Molecular cloning of the human angiotensin II type 2 receptor cDNA.

A human adult lung cDNA library was screened and one full-length human angiotensin II type 2 receptor (hAT2R) clone was isolated and characterized. The hAT2R cDNA clone contains a 1089-base-pair open reading frame which encodes a protein of 363 amino acid residues. The hAT2R is approximately 92% identical in sequence to the rat and mouse AT2R sequences. Specific binding of [125I]CGP42112A was demonstrated in membranes from COS-7 cells transiently transfected with the hAT2R cDNA. Scatchard analysis and ligand displacement profiles were typical of the AT2R. Northern analysis demonstrated that the hAT2R mRNA was abundantly expressed in human adult lung and in human fetal kidney. Additionally, the hAT2R mRNA was just detectable in human adult heart and aorta. In contrast, the rat AT2R mRNA was abundantly expressed in the rat brain and just detectable in the rat lung.

Adult↗