Specific And Efficient Transduction Of Cochlear Inner Hair Cells With Recombinant Adeno-associated Virus Type 3 Vector
adeno-associated virus; adenovirus; Biotechnology & Applied Microbiology; cochlea; ear; Experimental Medicine; gene transfer; gene transfer; generation; Genetics & Heredity; guinea-pig cochlea; hair cells; in-vivo; promoter; rat; Research &; serotype; therapy; transgene expression
Recombinant adeno-associated virus (AAV) vectors are of interest for cochlear gene therapy because of their ability to mediate the efficient transfer and long-term stable expression of therapeutic genes in a wide variety of postmitotic tissues with minimal vector-related cytotoxicity. In the present study, seven AAV serotypes (AAV1-5, 7, 8) were used to construct vectors. The expression of EGFP by the chicken P-actin promoter associated with the cytomegalovirus immediate-early enhancer in cochlear cells showed that each of these serotypes successfully targets distinct cochlear cell types. In contrast to the other serotypes, the AAV3 vector specifically transduced cochlear inner hair cells with high efficiency in vivo, while the AAV1, 2, 5, 7, and 8 vectors also transduced these and other cell types, including spiral ganglion and spiral ligament cells. There was no loss of cochlear function with respect to evoked auditory brain-stem responses over the range of frequencies tested after the injection of AAV vectors. These findings are of value for further molecular studies of cochlear inner hair cells and for gene replacement strategies to correct recessive genetic hearing loss due to monogenic mutations in these cells.
Liu Y H; Okada T; Sheykholeslami K; Shimazaki K; Nomoto T; Muramatsu S I; Kanazawa T; Takeuchi K; Ajalli R; Mizukami H; Kume A; Ichimura K; Ozawa K
Molecular Therapy
2005
2005-10
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1016/j.ymthe.2005.03.021" target="_blank" rel="noreferrer noopener">10.1016/j.ymthe.2005.03.021</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
Transcriptional regulation of the cholesterol 7 alpha-hydroxylase gene (CYP7A) by nuclear hormone receptors bound to the bile acid response elements (BARE)
activation; identification; promoter
Chiang J Y L; Stroup D; Crestani M; Sadeghpour A
1999
1999
Book/Monograph
n/a
Farnesoid X receptor responds to bile acids and represses cholesterol 7 alpha-hydroxylase gene (CYP7A1) transcription
orphan nuclear receptor; Biochemistry & Molecular Biology; expression; pathway; activation; identification; promoter; element; metabolites; hepg2 cells; ligands
Cholesterol 7 alpha-hydroxylase gene (CYP7A1) transcription is repressed by bile acids. The goal of this study is to elucidate the mechanism of CYP7A1 transcription by bile acid-activated farnesoid X receptor (FXR) in its native promoter and cellular context and to identify FXR response elements in the gene. In Chinese hamster ovary cells transfected with retinoid X receptor alpha (RXR alpha)/FXR, only chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) were able to stimulate a heterologous promoter/reporter containing an ecdysone response element. In HepG2 cells, all bile acids (25 mu M) were able to repress CYP7A1/luciferase reporter activity, and only CDCA and DCA further repressed reporter activity when cotransfected with RXR alpha/FXR, The concentration of CDCA required to inhibit 50% of reporter activity (IC(50)) was determined to be approximately 25 mu M without FXR and 10 mu M with FXR. Deletion analysis revealed that the bile acid response element located between nucleotides -148 and -128 was the FXR response element, but RXR alpha/FXR did not bind to this sequence. These results suggest that bile acid-activated FXR exerts its inhibitory effect on CYP7A1 transcription by an indirect mechanism, in contrast to the stimulation and binding of FXR to intestinal bile acid-binding protein gene promoter. Results also reveal that bile acid receptors other than FXR are present in HepG2 cells.
Chiang J Y L; Kimmel R; Weinberger C; Stroup D
Journal of Biological Chemistry
2000
2000-04
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1074/jbc.275.15.10918" target="_blank" rel="noreferrer noopener">10.1074/jbc.275.15.10918</a>
Regulation of cholesterol 7 alpha-hydroxylase gene (CYP7A1) transcription by the liver orphan receptor (LXR alpha)
bile acid synthesis; expression; Signaling; dietary-cholesterol; Bile acids; pathway; nuclear receptor; nuclear receptors; promoter; Genetics & Heredity; x-receptor; cytochrome P450; gene regulation; reverse cholesterol transport; hepg2 cells; coup-tfii; ligands
The cholesterol 7 alpha -hydroxylase gene (CYP7A1) plays an important role in regulation of bile acid biosynthesis and cholesterol homeostasis. Oxysterol receptor, LXR, stimulates, whereas the bile acid receptor, FXR, inhibits CYP7A1 transcription. The goal of this study was to investigate the role of LXR alpha on the regulation of rat, human and hamster CYP7A1 transcription in its native promoter and cellular context. Cotransfection with LXR alpha and RXR alpha expression plasmids strongly stimulated rat CYP7A1/luciferase reporter activity in HepG2 cells and oxysterol was not required. However, LXR alpha had much less effect on hamster and no significant effect on human CYP7A1 promoter activity in HepG2 cells. In Chinese hamster ovary cells, cotransfection with LXR alpha stimulated reporter activity by less than 2-fold and addition of 22(R)-hydroxycholesterol caused a small but significant stimulation of rat, human and hamster CYP7A1 promoter activity. At least two direct repeats of AGGTCA-like sequences with 4-base spacing (DR4) and five-base spacing (DR5), in previously identified bile acid response elements of the rat CYP7A1 were able to bind LXR alpha /RXR alpha and confer LXR alpha stimulation. However, LXR alpha did not bind to the corresponding sequences of the human gene and bound weakly to hamster and mouse DR4 sequences. Therefore, rats and mice have the unusual capacity to convert cholesterol to bile acids by LXR alpha -mediated stimulation of CYP7A1 transcription, whereas other species do not respond to cholesterol and develop hypercholesterolemia on a diet high in cholesterol. (C) 2001 Elsevier Science B.V. All rights reserved.
Chiang J Y L; Kimmel R; Stroup D
Gene
2001
2001-01
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1016/s0378-1119(00)00518-7" target="_blank" rel="noreferrer noopener">10.1016/s0378-1119(00)00518-7</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
Hepatocyte Growth Factor Family Negatively Regulates Hepatic Gluconeogenesis via Induction of Orphan Nuclear Receptor Small Heterodimer Partner in Primary Hepatocytes
signaling pathway; mice; hyperglycemia; Biochemistry & Molecular Biology; gene-expression; promoter; activated protein-kinase; insulin; diabetic-nephropathy; hepg2 liver-cells; upstream
Hepatic gluconeogenesis is tightly balanced by opposing stimulatory (glucagon) and inhibitory (insulin) signaling pathways. Hepatocyte growth factor (HGF) is a pleiotropic growth factor that mediates diverse biological processes. In this study, we investigated the effect of HGF and its family member, macrophage-stimulating factor (MSP), on hepatic gluconeogenesis in primary hepatocytes. HGF and MSP significantly repressed expression of the key hepatic gluconeogenic enzyme genes, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (Glc-6-Pase) and reduced glucose production. HGF and MSP activated small heterodimer partner (SHP) gene promoter and induced SHP mRNA and protein levels, and the effect of HGF and MSP on SHP gene expression was demonstrated to be mediated via activation of the AMP-activated protein kinase (AMPK) signaling pathway. We demonstrated that upstream stimulatory factor-1 (USF-1) specifically mediated HGF effect on SHP gene expression, and inhibition of USF-1 by dominant negative USF-1 significantly abrogated HGF-mediated activation of the SHP promoter. Elucidation of the mechanism showed that USF-1 bound to E-box-1 in the SHP promoter, and HGF increased USF-1 DNA binding on the SHP promoter via AMPK and DNA-dependent protein kinase-mediated pathways. Adenoviral overexpression of USF-1 significantly repressed PEPCK and Glc-6-Pase gene expression and reduced glucose production. Knockdown of endogenous SHP expression significantly reversed this effect. Finally, knockdown of SHP or inhibition of AMPK signaling reversed the ability of HGF to suppress hepatocyte nuclear factor 4 alpha-mediated up-regulation of PEPCK and Glc-6-Pase gene expression along with the HGF- and MSP-mediated suppression of gluconeogenesis. Overall, our results suggest a novel signaling pathway through HGF/AMPK/USF-1/SHP to inhibit hepatic gluconeogenesis.
Chanda D; Li T G; Song K H; Kim Y H; Sim J G; Lee C H; Chiang J Y L; Choi H S
Journal of Biological Chemistry
2009
2009-10
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1074/jbc.M109.022244" target="_blank" rel="noreferrer noopener">10.1074/jbc.M109.022244</a>
Fenofibrate Differentially Regulates Plasminogen Activator Inhibitor-1 Gene Expression via Adenosine Monophosphate-Activated Protein Kinase-Dependent Induction of Orphan Nuclear Receptor Small Heterodimer Partner
cells; binding; complex; fibrosis; Gastroenterology & Hepatology; promoter; beta; mechanisms; pai-1; shp; smad3
Plasminogen activator inhibitor type I (PAI-1) is a marker of the fibrinolytic system and serves as a possible predictor for hepatic metabolic syndromes. Fenofibrate, a peroxisome proliferator-activated receptor alpha (PPAR alpha) agonist, is a drug used for treatment of hyperlipidemia. Orphan nuclear receptor small heterodimer partner (SHP) plays a key role in transcriptional repression of crucial genes involved in various metabolic pathways. In this Study, we show that fenofibrate increased SHP gene expression in cultured liver cells and in the normal and diabetic mouse liver by activating the adenosine monophosphate-activated protein kinase (AMPK signaling pathway in a PPAR alpha-independent manner. Administration of transforming growth factor beta (TGF-beta) or a methionine-deficient and choline-deficient (MCD) diet to induce the progressive fibrosing steatohepatitis model in C57BL/6 mice was significantly reversed by fenofibrate via AMPK-mediated induction of SHP gene expression with a dramatic decrease in PAI-1 messenger RNA (mRNA) and protein expression along with other fibrotic marker genes. No reversal was observed in SHP null mice treated with fenofibrate. Treatment with another PPAR alpha agonist, WY14643, showed contrasting effects on these marker gene expressions in wild-type and SHP null mice, demonstrating the specificity of fenofibrate in activating AMPK signaling. Fenofibrate exhibited a differential inhibitory pattern on PAI-1 gene expression depending on the transcription factors inhibited by SHP. Conclusion: By demonstrating that a PPAR alpha-independent fenofibrate-AMPK-SHP regulatory cascade can play a key role in PAI-1 gene down-regulation and reversal of fibrosis, our study suggests that various AMPK activators regulating SHP might provide a novel pharmacologic option in ameliorating hepatic metabolic syndromes. (HEPATOLOGY 2009;50:880-892.)
Chanda D; Lee C H; Kim Y H; Noh J R; Kim D K; Park J H; Hwang J H; Lee M R; Jeong K H; Lee I K; Kweon G R; Shong M; Oh G T; Chiang J Y L; Choi H S
Hepatology
2009
2009-09
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1002/hep.23049" target="_blank" rel="noreferrer noopener">10.1002/hep.23049</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
Mechanisms of cholesterol and sterol regulatory element binding protein regulation of the sterol 12 alpha-hydroxylase gene (CYP8B1)
bile acids; bile-acid metabolism; Biochemistry & Molecular Biology; Biophysics; cholesterol; CYP8B1; gene regulation; liver; mice; mouse; pathway; promoter; rat; receptor lxr-alpha; SREBP; srebps; suppresses
Sterol 12alpha-hydroxylase (CYP8B1) is an obligatory enzyme for the synthesis of cholic acid and regulation of liver bile acid synthesis and intestine cholesterol absorption. The present study evaluates the roles for sterol regulatory element binding proteins (SREBPs) in the regulation of the CYP8B1 gene. Cholesterol feeding of mice and rats decreased the activity of CYP8B1, contrary to the up-regulation of cholesterol 7alpha-hydroxylase (CYP7A1). Cholesterol feeding also reduced mRNA levels for SREBP-1 but not for SREBP-2 in rat livers. Cholesterol and 25-hydroxycholesterol decreased the CYP8B1/luciferase reporter activity. Co-transfection of SREBP-1a and -1c stimulated CYP8B1 promoter activity, while SREBP-2 did not have any effects. Electrophoretic mobility shift assay and mutagenesis analyses identified several functional sterol regulatory elements (SRE) and E-box motifs in the rat CYP8B1 promoter. Our results indicate that SREBP-1a and -1c enhance transcription of the CYP8B1 gene through binding to SRE. Cholesterol loading reduces SREBP-1 mRNA expression in addition to reducing functional SREBP-1 protein, and results in decreasing CYP8B1 gene transcription. (C) 2004 Elsevier Inc. All rights reserved.
Yang Y Z; Eggertsen G; Gafvels M; Andersson U; Einarsson C; Bjorkhem I; Chiang J Y L
Biochemical and Biophysical Research Communications
2004
2004-08
Journal Article
<a href="http://doi.org/10.1016/j.bbrc.2004.06.069" target="_blank" rel="noreferrer noopener">10.1016/j.bbrc.2004.06.069</a>
On the mechanism of bile acid inhibition of rat sterol 12 alpha-hydroxylase gene (CYP8B1) transcription: roles of alpha-fetoprotein transcription factor and hepatocyte nuclear factor 4 alpha
activation; bile acid synthesise; Biochemistry & Molecular Biology; Biophysics; biosynthesis; Cell Biology; cholesterol 7-alpha-hydroxylase gene; Cyp7a1; cytochrome P450; expression; feedback-regulation; gene regulation; liver microsomal metabolism; nuclear receptor; promoter; receptor; repression
The sterol 12alpha-hydroxylase (CYP8B1) is a key enzyme of the bile acid biosynthetic pathway. It regulates the composition of bile acids in bile, i.e. ratio between cholic acid (CA) and chenodeoxycholic acid (CDCA). In similarity with cholesterol 7alpha-hydroxylase (CYP7A1), this enzyme is subjected to a negative feedback regulation by bile acids, It has been recently reported that bile acid-activated famesoid X receptor (FXR) induces the small heterodimer partner (SHP) that interacts with alpha-fetoprotein transcription factor (FTF) and down-regulates CYP7A1 transcription. We studied whether the same mechanism also regulated rat CYP8B1 gene transcription. Feeding rats with CDCA caused a 40-50% decrease of CYP8B1 and hepatocyte nuclear factor 4alpha (HNF4alpha) mRNA expression levels. This was associated with an increase in FTF mRNA expression, but SHP mRNA expression was not altered. Electrophoretic mobility shift assay (EMSA) and transient transfection assay of promoter/reporter genes coupled to mutagenesis analysis identified a putative bile acid response element (BARE) that has an HNF4alpha binding site embedded in two overlapping FTF binding sites. Mutation of the HNF4alpha binding site markedly reduced basal promoter activity and its repression by bile acids. Cotransfection with FTF strongly repressed CYP8B1 transcription. Interestingly, HNF4alpha could overcome the inhibitory effects of FTF and bile acids. We conclude that FTF and HNF4alpha not only play critical roles on CYP8B1 gene transcription, but also mediate bile acid feedback inhibition. This study reveals a novel mechanism by which bile acids inhibit rat CYP8B1 gene transcription by inducing FTF and inhibiting HNF4alpha expression. (C) 2002 Elsevier Science B.V. All rights reserved.
Yang Y Z; Zhang M; Eggertsen G; Chiang J Y L
Biochimica Et Biophysica Acta-Molecular and Cell Biology of Lipids
2002
2002-06
Journal Article
<a href="http://doi.org/10.1016/s1388-1981(02)00186-5" target="_blank" rel="noreferrer noopener">10.1016/s1388-1981(02)00186-5</a>