Fxr-activating Ligands Inhibit Rabbit Asbt Expression Via Fxr-shp-ftf Cascade
bile-acid transporter; cloning; down-regulation; Gastroenterology & Hepatology; identification; ileal; messenger-rna; negative feedback-regulation; nuclear receptor; Physiology; rats; real-time
The regulation of the rabbit apical sodium-dependent bile acid transporter ( ASBT) was studied both in vivo and in vitro. New Zealand White rabbits were fed 0.5% deoxycholic acid (DCA) or SC-435, a competitive ASBT inhibitor, for 1 wk. In DCA-fed rabbits, ASBT expression was repressed, associated with activated FXR, and evidenced by increased ileal short heterodimer partner (SHP) mRNA. Feeding SC-435 to the rabbits blocked bile acid absorption, decreased SHP mRNA, and increased ASBT expression. A 1.9-kb rabbit ASBT 5'-flanking region (promoter) was cloned, and a cis-acting element for alpha-fetoprotein transcription factor (FTF) was identified (-1166/-1158). The effects of transcriptional factors and different bile acids on the rabbit ASBT promoter were studied in Caco-2 cells. FTF stimulated the rabbit ASBT promoter activity fourfold but not after the FTF binding site was deleted from the promoter. Increasing the SHP protein notably inhibited FTF-dependent trans-activation of rabbit ASBT. Adding hydrophobic bile acids deoxycholic acid, chenodeoxycholic acid, and cholic acid, activating ligands for FXR, inhibited rabbit ASBT promoter activity in Caco-2 cells, but this inhibitory effect was abolished after the FTF binding site was deleted. Ursodeoxycholic acid and ursocholic acid, nonactivating ligands for FXR, did not repress ASBT promoter activity. Thus the rabbit ASBT promoter is negative-feedback regulated by bile acids via a functional FTF binding site. Only FXR-activating ligands can downregulate rabbit ASBT expression through the regulatory cascade FXR-SHP-FTF.
Li H; Chen F; Shang Q; Pan L X; Shneider B L; Chiang J Y L; Forman B M; Ananthanarayanan M; Tint G S; Salen G; Xu G R
American Journal of Physiology-Gastrointestinal and Liver Physiology
2005
2005-01
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1152/ajpgi.00170.2004" target="_blank" rel="noreferrer noopener">10.1152/ajpgi.00170.2004</a>
Variable Penetrance And Expressivity Of The Splice Altering 5t Sequence In The Cystic Fibrosis Gene
cftr gene; congenital bilateral absence; disease; Genetics & Heredity; males; messenger-rna; mutations; polymorphism; repeat; Research & Experimental Medicine; risk; vas-deferens
This manuscript reviews the frequencies, symptoms, testing, and reporting of genotypes with the 5T poly-thymidine tract which reduces splicing efficiency in the cystic fibrosis transmembrane conductance regulator ( CFTR) gene in congenital bilateral absence of the vas deferens (CBAVD) patients and in patients and fetuses with cystic fibrosis-like symptoms. The 5T sequence has not been included in the American College of Medical Genetics (ACMG) CFTR mutation panel recommended for screening pregnant women for an increased fetal risk of cystic fibrosis (CF; MIM 219700) because finding this allele would raise concern for possible CFTR gene-related symptoms in many fetuses, even though only a fraction inheriting 5T and another major CFTR mutation would develop CF-like symptoms. In contrast, 40-80% of the symptomatic patients with CBAVD (MIM 277180) are compound heterozygotes for the 5T sequence. This submission provides template report summaries for CBAVD patient results for the 5T allele when tested along with the 23 most common ACMG mutation panel. If CBAVD patients were also tested with the remaining 16 most common reported mutations in CBAVD, the derived proportion of patients with at least one CFTR mutant allele is predicted to increase from 63% to 97%. Testing for the 5T sequence in symptomatic patients and reflex 5T testing in fetuses found to carry a major CF allele are discussed because finding the 5T sequence in these patients lowers the risk of typical severe symptoms. Additional reflex testing for the number of TG repeats adjacent to a 5T allele further modifies the predicted long-term severity of disease symptoms in patients and fetuses that are compound heterozygotes for a major CF mutation and the 5T sequence. Even though patient advice can be modified currently based upon the adjacent TG-repeat number, the final most accurate risk frequencies with different 5T + TG-repeat alleles are likely to become available only after a larger patient population is completed with multiple well-defined clinical and mutation categories.
Lebo R V; Grody W W
Genetic Testing
2007
2007-03
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1089/gte.2006.9997" target="_blank" rel="noreferrer noopener">10.1089/gte.2006.9997</a>
Localization Of Nitric-oxide Synthase In The Mouse Olfactory And Vomeronasal System - A Histochemical, Immunological And In-situ Hybridization Study
accessory olfactory bulb; brain; messenger-rna; nadph-diaphorase activity; napdh diaphorase; napdh-p450 oxidoreductase; Neurosciences & Neurology; olfactory bulb; protein; reductase
The distribution of nitric oxide synthase (NOS) in the mouse olfactory bulb and olfactory epithelium, including the vomeronasal organ, was studied using an anti-NOS antibody, NADPH diaphorase histochemistry and in situ hybridization with NOS specific antisense oligonucleotide probes. Interneurons containing NOS protein and mRNA, and exhibiting NADPH diaphorase activity were detected in the plexiform layer of the main olfactory bulb and the granule cell layer of main and accessory olfactory bulbs. Periglomerular cells and granule cells in the main olfactory bulb were also NOS positive with diaphorase and immunostaining for NOS. In contrast, no evidence for NOS expression was found either in the main olfactory epithelium or in the vomeronasal organ, in spite of the strong diaphorase staining of the surface of the main olfactory epithelium. Polymerase chain reaction amplification experiments for detection of NOS gene expression further indicated that NOS is expressed in the olfactory bulb but not in either the main olfactory epithelium or vomeronasal organ. Use of an antibody raised against another enzyme, NADPH-P450 oxidoreductase, showed that this protein was strongly expressed in the olfactory epithelium, Activity of this enzyme may account for the diaphorase histochemical staining of the epithelia. An involvement of neuronal nitric oxide synthase in signalling in olfactory receptor neurons is therefore doubtful, although NOS is clearly expressed in neurons in both main and accessory olfactory bulbs.
Kishimoto J; Keverne E B; Hardwick J; Emson P C
European Journal of Neuroscience
1993
1993-12
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1111/j.1460-9568.1993.tb00236.x" target="_blank" rel="noreferrer noopener">10.1111/j.1460-9568.1993.tb00236.x</a>
Cholesterol 7-alpha-hydroxylase - Evidence For Transcriptional Regulation By Cholesterol Or Metabolic Products Of Cholesterol In The Rat
bile-acid synthesis; bile fistula; bile-acid synthesis; Biochemistry & Molecular Biology; biosynthesis; cloning; enterohepatic circulation; enzyme; hepatic cholesterol; hepatocytes; hmg-coa reductase; liver microsomes; lovastatin; messenger-rna; mevalonate; stimulation
Cholesterol 7alpha-hydroxylase, the rate-determining enzyme in the bile acid biosynthesis pathway, is regulated in a negative feedback manner by hydrophobic bile salts returning to the liver via the portal circulation. The role of cholesterol in the regulation of cholesterol 7alpha-hydroxylase and the interrelationship between the cholesterol and bile acid biosynthesis pathways remain controversial. The objective of the present study was to define the role of cholesterol in the regulation of cholesterol 7alpha-hydroxylase and determine the molecular level of its control. In order to avoid intestinal or intravenous administration of cholesterol, we manipulated the flow of cholesterol within the hepatocytes by decreasing cholesterol synthesis with lovastatin in bile fistula rats (bile acid synthesis is up-regulated), or by increasing cholesterol supply by administering mevalonate, a precursor of cholesterol, to rats with intact enterohepatic circulation (bile acid synthesis is normal). In the first series of studies, lovastatin was administered as a single intravenous bolus (10 mg/kg) to rats with chronic bile fistula and to rats with intact enterohepatic circulation (cholesterol and bile acid synthesis is normal). Three hours after lovastatin administration, cholesterol 7alpha-hydroxylase specific activity, enzyme mass, mRNA, and gene transcriptional activity were decreased by 35%, 32%, 56%, and 34%, respectively, in rats with chronic bile fistula. In rats with intact enterohepatic circulation, lovastatin administration resulted in a similar decrease (34%) of cholesterol 7alpha-hydroxylase specific activity. In the second group of experiments, rats with intact enterohepatic circulation were administered a 180 mum bolus of mevalonate followed by a continuous infusion of 180 mumol/h for 1.5, 3, 4.5, and 24 h prior to being killed. Continuous infusion of mevalonate increased cholesterol 7-alpha-hydroxylase specific activity, mRNA levels, and transcriptional activity by an average of 2- to 3-fold at all time intervals. We conclude that under circumstances in which cholesterol is present in excess, cholesterol 7alpha-hydroxylase transcriptional activity is up-regulated and removal of cholesterol from the hepatocytes is facilitated by an increase of bile acid synthesis. When cholesterol availability is decreased, cholesterol 7alpha-hydroxylase transcriptional activity is down-regulated leading to a decreased elimination of cholesterol via bile acid synthesis. In both instances, hepatic cholesterol homeostasis is effectively maintained.
Jones M P; Pandak W M; Heuman D M; Chiang J Y L; Hylemon P B; Vlahcevic Z R
Journal of Lipid Research
1993
1993-06
Journal Article or Conference Abstract Publication
n/a
Formation Of A Full Complement Of Cranial Proprioceptors Requires Multiple Neurotrophins
Anatomy & Morphology; brain-derived neurotrophic factor; central-nervous-system; Developmental Biology; expression; in-vivo; masseter muscle; mesencephalic trigeminal neurons; messenger-rna; mice; muscle spindles; muscle spindles; mutant; neurotrophin-3; neurotrophin-4; nt-3 activation; proprioceptive sensory neurons; sensory neurons; Trk receptors; Trk receptors
Fan G P; Copray S; Huang E J; Jones K; Yan Q; Walro J; Jaenisch R; Kucera J
Developmental Dynamics
2000
2000-06
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1002/(sici)1097-0177(200006)218:2%3C359::aid-dvdy9%3E3.0.co;2-l" target="_blank" rel="noreferrer noopener">10.1002/(sici)1097-0177(200006)218:2%3C359::aid-dvdy9%3E3.0.co;2-l</a>
Developmental And Genetic Influences Upon Gender Differences In Methamphetamine-induced Nigrostriatal Dopaminergic Neurotoxicity
bdnf mutant mice; brain; brain-derived neurotrophic factor (BDNF); differentiation; dopamine; estrogen; female mice; gonadal-hormones; messenger-rna; mptp-induced neurotoxicity; neurodegeneration; neuroprotection; neurotrophic factor; parkinsons-disease; sexual; sexual differences; testosterone; ventral mesencephalon
Dluzen D E; McDermott J L
Current Status of Drug Dependence / Abuse Studies: Cellular and Molecular Mechanisms of Drugs of Abuse and Neurotoxicity
2004
2004
Book Chapter
n/a
Bile Synthesis In Rat Models Of Inflammatory Bowel Diseases
acid synthesis; acute-phase response; acute-phase response; bile synthesis; cholesterol 7-alpha hydroxylase; cholesterol 7-alpha-hydroxylase gene; colitis; crohns-disease; experimental; General & Internal Medicine; inflammatory bowel disease; intestinal inflammation; messenger-rna; nitric-oxide; Research & Experimental Medicine; sulfonic-acid; ulcerative-colitis
Dikopoulos N; Schmid R M; Bachem M; Buttenschoen K; Adler G; Chiang J Y L; Weidenbach H
European Journal of Clinical Investigation
2007
2007-03
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1111/j.1365-2362.2007.01779.x" target="_blank" rel="noreferrer noopener">10.1111/j.1365-2362.2007.01779.x</a>
Transcriptional activation of the cholesterol 7 alpha-hydroxylase gene (CYP7A) by nuclear hormone receptors
rat; liver; bile acid synthesis; Biochemistry & Molecular Biology; expression; messenger-rna; elements; metabolism; promoter; nuclear; mutations; cytochrome P450; cholesterol 7; alpha-hydroxylase; bile acid response element; factor coup-tf; gene transcription and regulation; hormone receptor; retinoic acid receptors
The gene encoding cholesterol 7 alpha-hydroxylase (CYP7A), the rate-limiting enzyme in bile acid synthesis, is transcriptionally regulated by bile acids and hormones. Previously, we have identified two bile acid response elements (BARE) in the promoter of the CYP7A gene, The BARE II is located in nt -149/-118 region and contains three hormone response element (HRE)-like sequences that form two overlapping nuclear receptor binding sites. One is a direct repeat separated by one nucleotide DR1 (-146-TGGACTtAGTTCA-134) and the other is a direct repeat separated by five nucleotides DR5 (-139-AGTTCAaggccGGG TAA-123). Mutagenesis of these HRE sequences resulted in lower transcriptional activity of the CYP7A promoter/reporter genes in transient transfection assay in HepG2 cells. The orphan nuclear receptor, hepatocyte nuclear factor 4 (HNF-4)(1), binds to the DR1 sequence as assessed by electrophoretic mobility shift assay, and activates the CYP7A promoter/reporter activity by about 9-fold. Cotransfection of HNF-4 plasmid with another orphan nuclear receptor, chicken ovalbumin upstream promoter-transcription factor LI (COUP-TRI), synergistically activated the CYP7A transcription by 80-fold. The DR5 binds the RXR/RAR heterodimer, A hepatocyte nuclear factor-3 (HNF-3) binding site (-175-TGTTTGTTCT-166) was identified. HNF-3 was required for both basal transcriptional activity and stimulation of the rat CYP7A promoter activity by retinoic acid, Combinatorial interactions and binding of these transcription factors to BAREs may modulate the promoter activity and also mediate bile acid repression of CYP7A gene transcription.
Crestani M; Sadeghpour A; Stroup D; Galli G; Chiang J Y L
Journal of Lipid Research
1998
1998-11
Journal Article or Conference Abstract Publication
n/a
Nuclear receptor regulation of the human cholesterol 7 alpha-hydroxylase, sterol 27-hydroxylase and sterol 12 alpha-hydroxylase genes in bile acid synthesis
rat; biosynthesis; transcription factor; expression; hepatocytes; messenger-rna; pathway; activation; identification; cyp7a
Chiang J Y L; Chen W; Zhang M; Cowsley E; Yang Y Z
2001
2001
Book/Monograph
n/a
Nuclear receptor-mediated repression of human cholesterol 7 alpha-hydroxylase gene transcription by bile acids
liver; bile acid synthesis; Biochemistry & Molecular Biology; expression; messenger-rna; activation; identification; promoter; cytochrome P450; shp; cyp7a; hepg2 cells; mechanism of gene regulation; orphan receptor
Hydrophobic bile acids strongly repressed transcription of the human cholesterol 7 alpha -hydroxylase gene (CYP7A1) in the bile acid biosynthetic pathway in the Ever. Farnesoid X receptor (FXR) repressed CYP7A1/Luc reporter activity in a transfection assay in human liver-derived HepG2 cells, but not in human embryonic kidney (HEK) 293 cells. FXR-binding activity was required for bile acid repression of CYP7A1 transcription despite the fact that FXR did not bind to the CYP7A1 promoter. FXR-induced liver-specific factors must be required for mediating bile acid repression. Bile acids and FXR repressed endogenous CYP7A1 but stimulated a-fetoprotein transcription factor (FTF) and small heterodimer partner (SHP) mRNA expression in HepG2 cells. Feeding of rats with chenodeoxycholic acid repressed CYP7A1, induced FIT, but had no effect on SHP mRNA expression in the liver. FIT strongly repressed CYP7A1 transcription in a dose-dependent manner, and SHP further inhibited CYP7A1 in HepG2 cells, but not in HEK 293 cells. FXR only moderately stimulated SHP transcription, whereas FIT strongly inhibited SHP transcription in HepG2 cells. Results revealed that FTF was a dominant negative factor that was induced by bile acid-activated FXR to inhibit both CYP7A1 and SHP transcription. Differential regulation of FTF and SHP expression by bile acids may explain the wide variation in CYP7A1 expression and the rate of bile acid synthesis and regulation in different species.
Chen W L; Owsley E; Yang Y Z; Stroup D; Chiang J Y L
Journal of Lipid Research
2001
2001-09
Journal Article or Conference Abstract Publication
n/a
CHOLESTEROL 7-ALPHA-HYDROXYLASE IS UP-REGULATED BY THE COMPETITIVE INHIBITOR 7-OXOCHOLESTEROL IN RAT-LIVER
Biochemistry & Molecular Biology; expression; cloning; dietary-cholesterol; purification; messenger-rna; circadian-rhythm; reductase; microsomes; sequence; serum-cholesterol
Rats of the Sprague-Dawley strain were infused intravenously with a fat emulsion (Intralipid, trademark of Kabi Pharmacia, Uppsala, Sweden) containing 7-oxocholesterol. This resulted in an increased cholesterol 7alpha-hydroxylase activity in liver microsomes as compared to controls and was accompanied by increased levels of cholesterol 7alpha-hydroxylase mRNA and microsomal cholesterol 7alpha-hydroxylase protein. Rats were also fed a cholestyramine-supplemented diet and infused with 7-oxocholesterol. These animals excreted about half as much bile acids in faeces as cholestyramine-fed controls. Addition of 7-oxocholesterol to liver microsomes from normal rats in amounts corresponding to those present in microsomes from 7-oxocholesterol-treated rats inhibited the cholesterol 7alpha-hydroxylase activity by about 75%. Cholesterol induced a type-I binding spectrum when added to a purified bacterial-expressed cholesterol 7alpha-hydroxylase (P-450c7DELTA2-24). 7-Oxocholesterol competitively inhibited the cholesterol binding spectrum, while 7beta-hydroxycholesterol did not interfere with binding of cholesterol to the enzyme. It is concluded that treatment with the competitive inhibitor 7-oxocholesterol leads to a reduced bile acid biosynthesis and, as a consequence of reduced bile acid inhibition, a compensatory increase in cholesterol 7alpha-hydroxylase synthesis. The high enzyme activity measured in microsomal preparations from 7-oxocholesterol-treated rats may be due to a continuous conversion of 7-oxocholesterol into less inhibitory metabolites, e.g. 7beta-hydroxycholesterol. The latter compound was found in high concentrations in liver microsomes from rats treated with 7-oxocholesterol. The physiological importance of these results is discussed in relation to the previous findings that 7-oxocholesterol is accumulated in liver after cholesterol feeding and that 7-oxocholesterol is formed from cholesterol during lipid peroxidation.
Breuer O; Sudjanasugiaman E; Eggertsen G; Chiang J Y L; Bjorkhem I
European Journal of Biochemistry
1993
1993-08
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1111/j.1432-1033.1993.tb18082.x" target="_blank" rel="noreferrer noopener">10.1111/j.1432-1033.1993.tb18082.x</a>
Tamoxifen protects male mice nigrostriatal dopamine against methamphetamine-induced toxicity
disease; Pharmacology & Pharmacy; microglial activation; parkinsons-disease; Substantia nigra; messenger-rna; dopamine; transporter; neurotoxicity; Neuroprotection; estrogen-receptor-alpha; induced; striatum; gender-differences; estradiol; Parkinson's; striatal tissue fragments; Substantia nigra
The selective estrogen receptor modulator tamoxifen and estradiol were shown to protect nigrostriatal dopamine concentration loss by methamphetamine in female mice whereas male mice were protected only by tamoxifen. The present study examined the protective properties of tarnoxifen in male mice on several nigrostriatal dopaminergic markers and body temperature. Intact male mice were administered 12.5 or 50 mu g tarnoxifen 24 h before methamphetamine treatment. Basal body temperatures of male mice remained unchanged by the tarnoxifen treatment. Methamphetamine reduced striatal dopamine and its metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid concentrations, striatal and substantia nigra dopamine and vesicular monoamine transporter specific binding as well substantia nigra dopamine and vesicular monoamine transporter mRNA levels and increased striatal preproenkephalin mRNA levels. These methamphetamine effects were not altered by 12.5 mu g tarnoxifen except for increased striatal dopamine metabolites and turnover. Tamoxifen at 50 mu g reduced the methamphetamine effect on striatal dopamine concentration, dopamine transporter specific binding and prevented the increase in preproenkephalin mRNA levels; in the substantia nigra tamoxifen prevented the decrease of dopamine transporter mRNA levels. The present results show a tamoxifen dose-dependent prevention of loss of various dopaminergic markers against methamphetamine-induced toxicity in male mice. Since this is the only known hormonal protection of male mice against methamphetamine toxicity, these findings provide important new information on specific parameters of nigrostriatal dopaminergic function preserved by tarnoxifen. (C) 2007 Published by Elsevier Inc.
Bourque M; Liu B; Dluzen D E; Di Paolo T
Biochemical Pharmacology
2007
2007-11
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1016/j.bcp.2007.07.009" target="_blank" rel="noreferrer noopener">10.1016/j.bcp.2007.07.009</a>
INSITU HYBRIDIZATION STUDY OF OBESITY-ASSOCIATED ALTERATION IN GROWTH-HORMONE MESSENGER-RNA LEVELS
differentiation; Endocrinology & Metabolism; expression; fetal-rat; gene-transcription; glucocorticoid hormones; growth hormone; insitu hybridization; insulin; lean zucker rats; messenger-rna; Nutrition & Dietetics; pituitary-tumor cells; ribonucleic-acid; triiodothyronine
In order to investigate whether the impaired GH secretion associated with obesity is due to a pituitary disorder we studied GH mRNA levels by in situ hybridization in genetically obese and lean Zucker rats. The levels of GH mRNA were at least two fold lower in obese rats in comparison to that in lean controls as quantified by both the scanning of autoradiographs of tissue sections and Northern blot analysis. Quantification of somatotrophs revealed no significant difference in their number between lean and obese rat pituitaries. It is therefore likely that the attenuated GH mRNA levels in genetically obese Zucker rats are due to a decrease in GH transcripts per somatotroph rather than a result of a pituitary defect involving a preferential decrease in somatotroph population.
Ahmad I; Steggles A W; Finkelstein J A
International Journal of Obesity
1992
1992-06
Journal Article or Conference Abstract Publication
n/a
ALTERATION OF SOMATOSTATIN BUT NOT GROWTH HORMONE-RELEASING FACTOR PITUITARY BINDING-SITES IN OBESE ZUCKER RATS
binding site; cells; defect; Endocrinology & Metabolism; growth hormone-releasing factor; hypothalamus; messenger-rna; Physiology; pituitary; secretion; somatostatin; tissue; zucker rat
The present study was designed to determine whether the diminution of growth hormone (GH) secretion that occurs in obese Zucker rats is related to alterations of GH-releasing factor (GRF) or somatostatin (SRIF) pituitary binding sites. Cold saturation studies were performed in pituitary homogenates of 4-month-old lean and obese rats, using [I-125-Tyr-10]hGRF(1-44)NH-2 as radioligand and [I-127-Tyr-10]hGRF-(1-44)NH-2 as competitor, and in pituitary membrane preparations, using [I-125-Tyr-0, D-Trp-8]SRIF14 as radioligand and [I-127-Tyr-0, D-Trp-8]SRIF14 as competitor. In lean rats, analysis of the curves by the Ligand program revealed the presence of two distinct classes of GRF binding sites, the first being of high affinity (0.74 +/- 0.11 nM) and low capacity (118 +/- 31 fmol/mg protein), the second being of lower affinity (880 +/- 240 nM) and higher capacity (140 +/- 35 pmol/mg protein), and of a single class of SRIF binding sites (affinity: 0.40 +/- 0.12 nM; capacity: 24 +/- 6 fmol/mg protein). In obese rats, no difference was observed in GRF binding parameters for both classes of sites, but the concentration of somatostatin binding sites was reduced by 67% when compared to their lean littermates. These findings suggest that the SRIF pituitary receptors are down-regulated in obese Zucker rats and indicate that no alteration of GRF pituitary binding sites contribute to the blunted GH secretion observed in this model of obesity.
Abribat T; Finkelstein J A; Gaudreau P
Regulatory Peptides
1991
1991-10
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1016/0167-0115(91)90061-k" target="_blank" rel="noreferrer noopener">10.1016/0167-0115(91)90061-k</a>
Orphan receptors chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) and retinoid X receptor (RXR) activate and bind the rat cholesterol 7 alpha-hydroxylase gene (CYP7A)
arp-1; bile acids; Biochemistry & Molecular Biology; cloning; expression; liver; messenger-rna; regulatory protein-1; response elements; superfamily; thyroid-hormone
The cholesterol 7 alpha-hydroxylase gene (CYP7A) is transcriptionally regulated by a number of factors, including hormones, bile acids, and diurnal rhythm. Previous studies have identified a region from nucleotides (nt) -74 to -55 of the rat CYP7A promoter that enhanced bile acid repression of the SV40 early promoter, as assayed with a luciferase reporter gene in transiently transfected HepG2 cells. The rat CYP7A promoter/reporter activity was strongly stimulated by cotransfection with an expression plasmid encoding the nuclear hormone receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) in a dose-dependent manner. Site-directed mutagenesis in the region of nt -74 to -55 altered this stimulation. Recombinant COUP-TFII expressed in HepG2 or COS-1 cells were found to bind to nt -74 -55 and nt -149 -128 probes by electrophoretic mobility shift assay (EMSA) and by supershifting the corresponding band with COUP-TFII-specific antibodies. The region of nt -176 -117 was previously mapped as a retinoic acid response region and was found to bind retinoid X receptor (RXR). EMSA supershift assays of wild-type and mutant oligomers using antibody against RXR revealed that the sequences between nt -145 and -134 were important for RXR binding. We conclude that COUP-TFII stimulates the transcriptional activity of the rat CYP7A promoter by binding to the sequences between nt -74 to -54 and nt -149 to -128. RXR may stimulate CYP7A gene transcription by binding to a direct repeat of the hormone response element separated by one nucleotide located at nt -146 -134.
Stroup D; Crestani M; Chiang J Y L
Journal of Biological Chemistry
1997
1997-04
Journal Article
n/a
HNF4 and COUP-TFII interact to modulate transcription of the cholesterol 7 alpha-hydroxylase gene (CYP7A1)
bile-acid synthesis; bile acids; Biochemistry & Molecular Biology; chicken ovalbumin; cholesterol metabolism; dna-binding; hepatocyte nuclear factor 4; hepatocyte nuclear factor 4; hormone-receptor superfamily; messenger-rna; orphan receptors; promoter; receptors; response elements; retinoic acid; thyroid-hormone; transcriptional regulation; upstream promoter transcription factor II
The gene for cholesterol 7 alpha-hydroxylase (CYP7A1) contains a sequence at nt -149 to -118 that was found to play a large role in determining the overall transcriptional activity and regulation of the promoter. Hepatocyte nuclear factor 4 (HNF4) and chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) synergistically activate transcription of the CYP7A1 promoter, Transactivation of CYP7A1 by HNF4 in the human hepatoma cell line, HepG2, was enhanced by cotransfection with COUP-TFII or the basal transcription element binding protein (BTEB), HNF4 prepared from rat liver nuclear extracts bound to oligomers homologous to the nt -146 to -134 sequences in electrophoretic mobility shift assays (EMSA), which corresponded to a conserved region containing a direct repeat of hormone response elements spaced by one nucleotide (DR1), The sequences surrounding this DR1 were found to be essential for the HNF4 transactivation. In vitro-translated COUP-TFII was found to bind the adjacent sequences from nt -139 to -128 (DRO), but COUP-TFII interacted with this region at a much lower affinity than to the COUP-TFII-site at nt -72 to -57 (DR4), Mutations at nt -139 to -128 or nt -72 to -57 reduced the COUP-TFII and HNF4 synergy; however, these COUP-TFII-binding sequences were not absolutely required for the cooperative effect of HNF4 and COUP-TFII on transactivation. These results indicated that the observed transactivation was the result of protein/protein interactions facilitated by the juxtaposition of the binding elements.
Stroup D; Chiang J Y L
Journal of Lipid Research
2000
2000-01
Journal Article
n/a
BILE-ACID SYNTHESIS .6. REGULATION OF CHOLESTEROL 7-ALPHA-HYDROXYLASE BY TAUROCHOLATE AND MEVALONATE
3-hydroxy-3-methylglutaryl; 7-alpha-hydroxylase; bile acids; Biochemistry & Molecular Biology; biosynthesis; circadian-rhythm; cloning; coenzyme; enzyme; hepatocytes; hmg-coa reductase; liver; messenger-rna; rat-liver microsomes; reductase; substrate
Taurocholate, a relatively hydrophobic bile salt, is a potent down-regulator of HMG-CoA reductase and cholesterol 7-alpha-hydroxylase (C7-alpha-H), the rate-determining enzymes of the cholesterol and bile acid biosynthetic pathways, respectively. Inhibition of cholesterol synthesis with a bolus dose of mevinolin (lovastatin) a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, profoundly decreases the specific activity of cholesterol 7-alpha-hydroxylase and rate of bile acid synthesis in rats with complete biliary diversion. It is therefore conceivable that taurocholate may suppress cholesterol 7-alpha-hydroxylase primarily by down-regulating the activity of HMG-CoA reductase. To test this hypothesis, taurocholate was coinfused simultaneously to rats with chronic bile fistula with mevalonate (administered as mevalonolactone), an intermediate in the cholesterol biosynthetic pathway. Mevalonolactone was administered to provide a constant supply of newly synthesized cholesterol to cholesterol 7-alpha-hydroxylase, in order to overcome any inhibitory effect of taurocholate on HMG-Coa reductase. Infusions were started 72 h after biliary diversion, and carried out for an additional 48 h. Complete biliary diversion resulted in an increase in C7-alpha-H specific activity (510%), protein mass (550%), steady-state mRNA levels (1430%), and transcriptional activities (330%) as compared to control rats with intact enterohepatic circulations. When rats with biliary diversion were infused intraduodenally with taurocholate, the specific activities of HMG-CoA reductase and cholesterol 7-alpha-hydroxylase activities decreased by 75% (P < 0.001) and 73% (P < 0.001), respectively. Cholesterol 7-alpha-hydroxylase mass, mRNA, and transcriptional activity decreased after intraduodenal infusion of taurocholate to levels similar to those of rats with an intact enterohepatic circulation. The combination of constant infusion of mevalonate and taurocholate failed to reverse the inhibitory effects of taurocholate on cholesterol 7-alpha-hydroxylase activity, mRNA levels, and in vitro transcriptional rates. These data provide evidence that taurocholate represses cholesterol 7-alpha-hydroxylase at the level of gene transcription, and not via down-regulation of HMG-CoA reductase. Infusion of mevalonate alone to biliary diverted rats did not alter cholesterol 7-alpha-hydroxylase activity or mRNA levels, while leading to a 57% decrease in C7-alpha-H gene transcription. This latter finding suggests that mevalonate or its metabolites may be capable of stabilizing C7-alpha-H mRNA levels while down-regulating transcriptional activity.
Pandak W M; Vlahcevic Z R; Chiang J Y L; Heuman D M; Hylemon P B
Journal of Lipid Research
1992
1992-05
Journal Article
n/a
Identification of Multiple Metabolic Enzymes from Mice Cochleae Tissue Using a Novel Functional Proteomics Technology
Animal tissues; Beef; Biology; cancer; cancer metabolism; cell metabolism; Cochlea; draft; Drug therapy; E coli; Electrophoresis; Elution; Enzymatic activity; Enzyme assays; Enzyme metabolism; Enzymes; Feasibility studies; Functional analysis; Gel electrophoresis; Genes; Genomics; Hearing impairment; Kinases; Mass spectrometry; Mass spectroscopy; medicine; messenger-rna; metabolism; mice; NAD(P)H oxidase; NADH; Neurobiology; Neurosciences; Nicotinamide adenine dinucleotide; Noise; Otolaryngology; Oxidation-reduction reactions; Phosphatase; Protein expression; proteins; Proteomes; Proteomics; Science & Technology - Other Topics; Sciences: Comprehensive Works; Scientific imaging; Studies; Substance abuse treatment; Technology; United States--US
A new type of technology in proteomics was developed in order to separate a complex protein mixture and analyze protein functions systematically. The technology combines the ability of two-dimensional gel electrophoresis (2-DE) to separate proteins with a protein elution plate (PEP) to recover active proteins for functional analysis and mass spectrometry (MS)-based identification. In order to demonstrate the feasibility of this functional proteomics approach, NADH and NADPH-dependent oxidases, major redox enzyme families, were identified from mice cochlear tissue after a specific drug treatment. By comparing the enzymatic activity between mice that were treated with a drug and a control group significant changes were observed. Using MS, five NADH-dependent oxidases were identified that showed highly altered enzymatic activities due to the drug treatment. In essence, the PEP technology allows for a systematic analysis of a large enzyme family from a complex proteome, providing insights in understanding the mechanism of drug treatment.
Wang D L; Li H; Liang R Q; Bao J X
PLOS ONE
2015
2015-03
Journal Article
<a href="http://doi.org/10.1371/journal.pone.0121826" target="_blank" rel="noreferrer noopener">10.1371/journal.pone.0121826</a>
An overlapping binding site in the CYP7A1 promoter allows activation of FXR to override the stimulation by LXR alpha
7-alpha-hydroxylase gene; activity; bile-acid biosynthesis; cholesterol; cholesterol 7 alpha-hydroxylase; dietary-cholesterol; down-regulation; farnesoid X; fetoprotein transcription factor; Gastroenterology & Hepatology; heterodimer partner; liver X receptor; LXR binding site; messenger-rna; metabolism; orphan nuclear receptor; Physiology; rat; receptor; regulation; short; transcriptional; x-receptor
An overlapping binding site in the CYP7A1 promoter allows activation of FXR to override the stimulation by LXR alpha. Am J Physiol Gastrointest Liver Physiol 293: G817-G823, 2007. First published August 9, 2007; doi:10.1152/ajpgi.00209.2007.-The aim of this study was to explore why in rabbits activation of farnesoid X receptor (FXR) is dominant over activated liver X receptor-alpha (LXR alpha) in the regulation of CYP7A1. We cloned the rabbit CYP7A1 promoter and found a fetoprotein transcription factor (FTF) binding element embedded within the LXR alpha binding site (LXRE). Gel shift assays demonstrated that FTF competes with LXR alpha for binding to LXRE. Short heterodimer partner (SHP) enhances the competitive ability of FTF. Studies in HepG2 cells showed that SHP combined with FTF had more powerful effect to offset the stimulation of CYP7A1 by LXR alpha. Gel shift and chromatin immunoprecipitation assays demonstrated that SHP with FTF diminished LXR alpha\binding to the CYP7A1 promoter. In vivo studies in rabbits fed cholesterol for 10 days showed that hepatic expression of SHP but not FTF rose and LXR alpha-bound LXRE decreased. We propose that the SHP/FTF heterodimer occupies LXRE via the embedded FTF binding element and blocks LXR alpha from recruiting to LXRE. Therefore, activation of FXR, which upregulates SHP expression, will eliminate the stimulatory effect of LXR alpha on the CYP7A1 promoter because increased levels of SHP combined with FTF diminish the recruitment of LXR alpha to CYP7A1 promoter.
Shang Q; Pan L X; Saumoy M; Chiang J Y L; Tint G S; Salen G; Xu G R
American Journal of Physiology-Gastrointestinal and Liver Physiology
2007
2007-10
Journal Article
<a href="http://doi.org/10.1152/ajpgi.00209.2007" target="_blank" rel="noreferrer noopener">10.1152/ajpgi.00209.2007</a>
Lung edema clearance: 20 years of progress selected contribution: Long-term effects of beta(2)-adrenergic receptor stimulation on alveolar fluid clearance in mice
acute lung injury; alveolar; beta-adrenergic-receptor; desensitization; down-regulation; epithelium; hydrostatic pulmonary-edema; in-vivo; liquid clearance; lung fluid balance; messenger-rna; Physiology; pulmonary edema; rat lung; resolution; sodium transport; Sport Sciences
Stimulation of active fluid transport with beta-adrenergic receptor (betaAR) agonists can accelerate the resolution of alveolar edema. However, chronic betaAR-agonist administration may cause betaAR desensitization and downregulation that may impair physiological responsiveness to betaAR-agonist stimulation. Therefore, we measured baseline and terbutaline- (10(-3) M) stimulated alveolar fluid clearance in mice that received subcutaneously (miniosmotic pumps) either saline or albuterol (2 mg.kg(-1).day(-1)) for 1, 3, or 6 days. Continuous albuterol administration increased plasma albuterol levels (10(-5) M), an effect that was associated with 1) a significant decrease in betaAR density and 2) attenuation, but not ablation, of maximal terbutaline- induced cAMP production. Forskolin-mediated cAMP-release was unaffected. Continuous albuterol infusion stimulated alveolar fluid clearance on day 1 but did not increase alveolar fluid clearance on days 3 and 6. However, terbutaline- stimulated alveolar fluid clearance in albuterol-treated mice was not reduced compared with saline-treated mice. Despite significant reductions in betaAR density and agonist-mediated cAMP production by long-term betaAR-agonist exposure, maximal betaAR-agonist-mediated increase in alveolar fluid clearance is not diminished in mice.
Sartori C; Fang X; McGraw D W; Koch P; Snider M E; Folkesson H G; Matthay M A
Journal of Applied Physiology
2002
2002-11
Journal Article
<a href="http://doi.org/10.1152/japplphysiol.00275.2002" target="_blank" rel="noreferrer noopener">10.1152/japplphysiol.00275.2002</a>
Development of fusimotor innervation correlates with group Ia afferents but is independent of neurotrophin-3
deficits; Developmental Biology; expression; fusimotor innervation; growth factor; messenger-rna; mice; motor-neurons; muscle spindle; nervous-system; Neurosciences & Neurology; neurotrophin-3; proprioception; rat; transgenic mice
Fusimotor neurons, group Ia afferents and muscle spindles are absent in mutant mice lacking the gene for neurotrophin-3 (NT3). To partition the effect of Ia afferent or spindle absence from that of NT3 deprivation on fusimotor neuron development, we examined the fusimotor system in a mutant mouse (NesPIXpNT3) that lacks Ia afferents and spindles, but has normal or elevated tissue levels of NT3 during embryogenesis. Fusimotor fibers were absent in lumbar ventral spinal roots, and limb muscles were devoid of Ia afferents and spindles in adult NesPIXpNT3 mice. In contrast, no deficiency in motoneuron numbers was observed in the trigeminal nucleus which contains cell bodies of motor axons innervating muscles of mastication. Spindles and Ia afferents were also present in the masticatory muscles. Thus, the development and/or survival of fusimotor neurons correlates with the presence of Ia afferents and/or spindles, and not with the amount of NT3 in the spinal cord or muscle. (C) 1998 Elsevier Science B.V. All rights reserved.
Ringstedt T; Copray S; Walro J; Kucera J
Developmental Brain Research
1998
1998-12
Journal Article
<a href="http://doi.org/10.1016/s0165-3806(98)00146-1" target="_blank" rel="noreferrer noopener">10.1016/s0165-3806(98)00146-1</a>
Transcriptional suppression of cytochrome P450 genes by endogenous and exogenous chemicals
bile acid; cholesterol 7 alpha-hydroxylase; cyp2c11 gene; cyp7a1 transcription; down-regulation; messenger-rna; nuclear receptor; Pharmacology & Pharmacy; polycyclic aromatic-hydrocarbons; pregnane X receptor; rat-liver
This article is an invited report of a symposium sponsored by the Division for Drug Metabolism of the American Society for Pharmacology and Experimental Therapeutics held at Experimental Biology 2003 in San Diego, California, April 11 - 15, 2003. Several members of the cytochrome P450 (P450) superfamily are induced after exposure to a variety of chemical signals, and we have gained considerable mechanistic insight into these processes over the past four decades. In addition, the expression of many P450s is suppressed in response to various endogenous and exogenous chemicals; however, relatively little is known about the molecular mechanisms involved. The goal of this symposium was to critically examine our current understanding of molecular mechanisms involved in transcriptional suppression of CYP genes by endogenous and exogenous chemicals. Specific examples were drawn from the following chemical categories: polycyclic and halogenated aromatic hydrocarbon environmental toxicants, inflammatory mediators, the endogenous sterol dehydroepiandrosterone and peroxisome proliferators, and bile acids. Multiple molecular mechanisms are involved in transcriptional suppression, and these processes often involve rather complex cascades of transcription factors and other regulatory proteins. Mechanistic studies of CYP gene suppression can enhance our understanding of how organisms respond to xenobiotics as well as to perturbations in endogenous chemicals involved in maintaining homeostasis.
Riddick D S; Lee C; Bhathena A; Timsit Y E; Cheng P Y; Morgan E T; Prough R A; Ripp S L; Miller K K M; Jahan A; Chiang J Y L
Drug Metabolism and Disposition
2004
2004-04
Journal Article
<a href="http://doi.org/10.1124/dmd.32.4.367" target="_blank" rel="noreferrer noopener">10.1124/dmd.32.4.367</a>
Repeated stressor exposure regionally enhances beta-adrenergic receptor-mediated brain IL-1 beta production
Anhedonia; antidepressant treatments; Chronic mild stress; chronic psychosocial stress; Cytokine; depression; depressive-like behavior; hippocampal neurogenesis; Immunology; inflammatory cytokines; ligand-binding; messenger-rna; Neurosciences & Neurology; Norepinephrine; paraventricular nucleus; prefrontal cortex; Psychiatry; rat; rat-brain; Receptor binding; Sensitization
It has been proposed that increased brain cytokines during repeated stressor exposure can contribute to neuropathological changes that lead to the onset of depression. Previous studies demonstrate that norepinephrine acting via beta-adrenergic receptors (beta-ARs) mediate brain IL-1 production during acute stressor exposure. The aim of the current studies was to examine how the regulation of brain cytokines by adrenergic signaling might change following repeated stressor exposure. Fischer rats were exposed to four days of chronic mild stress and 24 h after the last stressors beta-AR expression, norepinephrine turnover, and beta-AR-mediated induction of brain IL-1 were measured in limbic areas (e.g. hypothalamus, hippocampus, amygdala, and prefrontal cortex) and brainstem. Repeated stressor exposure resulted in decreases in beta-AR expression (B-max) measured by saturation binding curves in many limbic brain areas, while an increase was observed in the brainstem. This coincided with significant increases in norepinephrine turnover in the prefrontal cortex, hypothalamus, and amygdala, a significant increase in norepinephrine turnover was not observed in the hippocampus or brainstem. Stress increased overall IL-1 production in the amygdala (both basal and stimulated). While stress did not affect basal IL-1 levels in any other brain area, central administration of isoproterenol (a beta-AR agonist) augmented IL-1 production in the hypothalamus of stressed animals. These data indicate that repeated stressor exposure results in brain area specific enhancements in beta-AR-mediated IL-1 production and extends current knowledge of stress-induced enhancement of brain cytokine beyond sensitized response to immunological stimuli. (C) 2012 Elsevier Inc. All rights reserved.
Porterfield V M; Gabella K M; Simmons M A; Johnson J D
Brain Behavior and Immunity
2012
2012-11
Journal Article
<a href="http://doi.org/10.1016/j.bbi.2012.08.001" target="_blank" rel="noreferrer noopener">10.1016/j.bbi.2012.08.001</a>
Hep G2 cells: A model for studies on regulation of human cholesterol 7 alpha-hydroxylase at the molecular level
bile acid; bile-acid synthesis; biosynthesis; density-lipoprotein receptors; expression; Gastroenterology & Hepatology; hepatocytes; line; liver; messenger-rna; metabolism; monolayer-cultures; Physiology; primary; rat; transcriptional activity
The present study examines the feedback control governing human cholesterol 7 alpha-hydroxylase mRNA expression in the human hepatoblastoma cell line, Hep G2. Glycochenodeoxycholate (GCDC) and glycodeoxycholate, hydrophobic bile salts, decreased cholesterol 7 alpha-hydroxylase mRNA levels and bile acid synthesis in a concentration-dependent (76 +/- 8%, P < 0.001, and 48 +/- 3%, P < 0.01, respectively) and time-dependent manner. Cholesterol 7 alpha-hydroxylase mRNA levels were repressed with a half-maximal inhibitory concentration of < 12.5 mu M by GCDC and a half-life of 30 min by 100 mu M of the bile acid. The addition of actinomycin D (10 mu g/ml) alone or in combination with GCDC (100 mu M) led to similar concentration- and time-dependent suppression of cholesterol 7 alpha-hydroxylase mRNA. Glycocholate (100 mu M), not internalized based on lack of uptake of a fluorescent cholate analogue, had no effect on cholesterol 7 alpha-hydroxylase mRNA or total bile acid synthesis. In cultures transfected with a rat cholesterol 7 alpha-hydroxylase promoter construct, reporter gene activity was decreased (31%, P < 0.01) by GCDC (100 alpha M). Hep G2 cells maintain the intracellular machinery to express and rapidly regulate human cholesterol 7 alpha-hydroxylase by hydrophobic bile acids. These data suggest that Hep G2 cells will support functional studies of the human cholesterol 7 alpha-hydroxylase gene.
Pandak W M; Stravitz R T; Lucas V; Heuman D M; Chiang J Y L
American Journal of Physiology-Gastrointestinal and Liver Physiology
1996
1996-03
Journal Article
<a href="http://doi.org/10.1152/ajpgi.1996.270.3.g401" target="_blank" rel="noreferrer noopener">10.1152/ajpgi.1996.270.3.g401</a>
HORMONAL-CONTROL OF GROWTH IN THE GENETICALLY-OBESE ZUCKER RAT .1. LINEAR GROWTH, PLASMA INSULIN-LIKE GROWTH FACTOR-I (IGP-I) AND IGF-BINDING PROTEINS
body-composition; developmental-changes; Endocrinology & Metabolism; expression; gh; hepatocytes; messenger-rna; radioimmunoassay; secretion; serum; somatomedin-c
The genetically obese Zucker rat is a widely used model of early-onset obesity. Like obese children, these obese rats are hyperinsulinemic and have low GH secretion. However, data on linear growth and insulin-like growth factor-I (IGF-I) levels in this model are scanty and contradictory. In the present study, we investigated linear growth and its hormonal control in Zucker rats (male and female) from 4-20 weeks of age. In the obese animals, compared to their lean littermates, the naso-anal length was normal or slightly greater, whereas the tails and femurs were shorter. The plasma concentration of IGF-I increased between 4-20 weeks of age, and IGF-I levels were normal or slightly higher in the obese animals. The serum level of IGF-binding protein-3 (IGFBP-3) measured by Western ligand blotting was not significantly different in lean vs, obese rats. To assess the IGF-I response to GH, bovine GH was administered (250 mu g/100 g BW, ip, daily for 3 days) to 16- to 20-week-old female Zucker rats; plasma IGF-I concentrations increased more in the obese (percent increase over baseline, 347 +/- 44% vs. 194 +/- 31%; P < 0.01). These results show that despite low GH secretion, genetically obese Zucker rats have 1) normal linear (nasoanal) growth, 2) normal or increased circulating levels of IGF-I and IGFBP-3, and 3) increased plasma IGF-I responses to exogenous GH. These results suggest that the GH-independent growth in this model could result from direct effects of hyperinsulinism on circulating IGF-I and IGFBP-3 levels and/or indirect effects through increased GH receptor function.
Nguyenyamamoto L; Deal C L; Finkelstein J A; Vanvliet G
Endocrinology
1994
1994-03
Journal Article
<a href="http://doi.org/10.1210/en.134.3.1382" target="_blank" rel="noreferrer noopener">10.1210/en.134.3.1382</a>