Structure and functions of human oxysterol 7alpha-hydroxylase cDNAs and gene CYP7B1.
Humans; Animals; Mice; Cell Line; Transfection; Base Sequence; Molecular Sequence Data; Chromosome Mapping; Cytochrome P450 Family 7; DNA; Luciferases/genetics; Cytochrome P-450 Enzyme System/*genetics/metabolism; Steroid Hydroxylases/*genetics/metabolism; Hydroxycholesterols/metabolism; Codon; Northern; Blotting; Transcription; Genetic; Cloning; Molecular; Genetic/genetics; Promoter Regions; Nucleic Acid; Complementary/biosynthesis/*isolation & purification; Initiator; Regulatory Sequences
Oxysterol 7alpha-hydroxylase has broad substrate specificity for sterol metabolites and may be involved in many metabolic processes including bile acid synthesis and neurosteroid metabolism. The cloned human oxysterol 7alpha-hydroxylase (CYP7B1) cDNA encodes a polypeptide of 506 amino acid residues that shares 40% sequence identity to human cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in the conversion of cholesterol to bile acids in the liver. In contrast to the liver-specific expression of CYP7A1, CYP7B1 mRNA transcripts were detected in human tissues involved in steroid genesis (brain, testes, ovary, and prostate) and in bile acid synthesis (liver) and reabsorption (colon, kidney, and small intestine). The human oxysterol 7alpha-hydroxylase transiently expressed in 293/T cells was able to catalyze 7alpha-hydroxylation of
Wu Z; Martin K O; Javitt N B; Chiang J Y
Journal of lipid research
1999
1999-12
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
HNF4 and COUP-TFII interact to modulate transcription of the cholesterol 7alpha-hydroxylase gene (CYP7A1).
Humans; Animals; Rats; Liver/metabolism; Recombinant Proteins/metabolism; Base Sequence; Mutation; DNA Primers; Cholesterol 7-alpha-Hydroxylase/*genetics; Transcription Factors/*metabolism; Hepatocyte Nuclear Factor 4; DNA-Binding Proteins/*metabolism; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Luciferases/genetics; COUP Transcription Factor II; COUP Transcription Factors; Phosphoproteins/*metabolism; Genes; Sprague-Dawley; Cultured; Genetic; Tumor Cells; Reporter; Promoter Regions; *Transcription; *Receptors; Steroid
The gene for cholesterol 7alpha-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 (DR0), 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
Stroup D; Chiang J Y
Journal of lipid research
2000
2000-01
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Transcriptional activation of the cholesterol 7alpha-hydroxylase gene (CYP7A) by nuclear hormone receptors.
Animals; Rats; Transcription Factors/metabolism; Base Sequence; Molecular Sequence Data; DNA/metabolism; Cholesterol 7-alpha-Hydroxylase/*genetics; Hepatocyte Nuclear Factor 4; Mutagenesis; Luciferases/genetics; Retinoid X Receptors; Phosphoproteins/metabolism; COUP Transcription Factor II; COUP Transcription Factors; *Transcriptional Activation; Bile Acids and Salts/biosynthesis; DNA-Binding Proteins/metabolism; Hormones/*physiology; Oligonucleotide Probes/metabolism; Genes; Cultured; Receptors; Genetic; Cytoplasmic and Nuclear/*physiology; Tumor Cells; Reporter; Retinoic Acid/metabolism; Promoter Regions; Nucleic Acid; Site-Directed; *Receptors; Repetitive Sequences; Steroid
The gene encoding cholesterol 7alpha-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 II (COUP-TFII), 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
Journal of lipid research
1998
1998-11
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Hormonal regulation of the cholesterol 7 alpha-hydroxylase gene (CYP7).
Animals; Rats; Gene Expression Regulation; Cell Count; Transfection; Base Sequence; Molecular Sequence Data; Phorbol Esters/pharmacology; Cholesterol 7-alpha-Hydroxylase/*genetics; Luciferases/genetics; Cyclic AMP/pharmacology; Hormones/*pharmacology; Second Messenger Systems/physiology; Cultured; Genetic; Tumor Cells; Cloning; Molecular; *Promoter Regions; *Transcription; Enzymologic/*physiology
The transcriptional regulation of the rat cholesterol 7 alpha-hydroxylase gene (CYP7) by hormones and signal transduction pathways was studied by transient transfection assay of the promoter activity. HepG2 cells were transfected with deletion mutants of the CYP7 upstream region linked to the luciferase reporter gene. The transcription of CYP7/luciferase chimeric genes was higher in confluent than in subconfluent cultures of HepG2 cells. Glucocorticoid receptors, in the presence of dexamethasone, up-regulated the CYP7 gene through two regions located between -3262 and -2803, and between -344 and -222, respectively. Thyroid hormones did not have any effect on the promoter activity. Insulin inhibited the promoter activity through sequences located between -344 and -222, and abolished the stimulation by dexamethasone. Hence, the insulin effect was dominant over that of glucocorticoids. Treatment of transfected HepG2 cells with phorbol
Crestani M; Stroup D; Chiang J Y
Journal of lipid research
1995
1995-11
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Identification and characterization of a putative bile acid-responsive element in cholesterol 7 alpha-hydroxylase gene promoter.
Humans; Animals; Rats; Gene Expression Regulation; Base Sequence; Bile Acids and Salts/*metabolism; Cholesterol 7-alpha-Hydroxylase/*genetics/metabolism; Molecular Sequence Data; Recombinant Fusion Proteins/genetics/metabolism; Luciferases/genetics; DNA-Binding Proteins/metabolism; Deoxyribonuclease I; Nuclear Proteins/metabolism; Oligodeoxyribonucleotides; Thyroid Hormones/genetics; Sprague-Dawley; Genetic; Enzymologic; Cloning; Molecular; *Promoter Regions; Antigens; Polyomavirus Transforming/genetics
Nucleotide sequences of a 7997-base pair SacI fragment spanning 3643 base pairs of the upstream promoter region to exon 4 of the rat cholesterol 7 alpha-hydroxylase gene (CYP7) have been determined. DNase I footprinting and electrophoretic mobility shift assay of the proximal promoter from nucleotides -346 to +36 revealed two protected regions which specifically shifted proteins in rat liver nuclear extracts. Footprint A (nucleotides -81 to -35) contained a cluster of overlapping sequence motifs of TGT3, steroid/thyroid hormone response elements (7 alpha TRE), hepatocyte nuclear factors 1 and 4, and CAAT/enhancer-binding protein alpha and has been shown to confer bile acid repression of the CYP7 gene promoter activity. Footprint B (nucleotides -148 to -129) contained a sequence motif HNF4. When footprint A (-101 to -49) or 7 alpha TRE (-73 to -55) sequence was linked upstream to a heterologous SV40 promoter/luciferase plasmid and transiently transfected into HepG2 cells, taurodeoxycholate suppressed the SV40 promoter activity. Electrophoretic mobility shift assays revealed that one or two bands shifted by the 7 alpha TRE or by a direct repeat sequence in 7 alpha TRE were absent when liver nuclear extracts of deoxycholic acid-treated rats were used. Similar gel shift patterns were also observed when human 7 alpha TRE or human liver nuclear extracts were used. The rat direct repeat sequence interacted with two polypeptides (M(r) = 57,000 and 116,000) in both rat and human liver nuclear extracts. These results suggest that hydrophobic bile acids may suppress the CYP7 gene expression by binding to a bile acid receptor which interacts with and prevents the binding of liver nuclear protein(s) to a bile acid-responsive element and that the core of bile acid-responsive element is a direct repeat.
Chiang J Y; Stroup D
The Journal of biological chemistry
1994
1994-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Nuclear receptor-mediated repression of human cholesterol 7alpha-hydroxylase gene transcription by bile acids.
Humans; Animals; Rats; Cell Line; Transfection; Liver/metabolism; Reverse Transcriptase Polymerase Chain Reaction; DNA/metabolism; CHO Cells; Cricetinae; Cholesterol 7-alpha-Hydroxylase/*genetics; Bile Acids and Salts/*pharmacology; *Membrane Glycoproteins; *Hydroxysteroid Dehydrogenases; Caco-2 Cells; Carrier Proteins/genetics/physiology; DNA-Binding Proteins/drug effects/genetics/physiology; Gene Expression/*drug effects; Kidney; Luciferases/genetics; Recombinant Fusion Proteins/metabolism; Retinoid X Receptors; Taurocholic Acid/pharmacology; Transcription Factors/drug effects/genetics/physiology; Cultured; Receptors; RNA; Genetic/drug effects; Messenger/analysis; Transcription; Genetic; Tumor Cells; Promoter Regions; Embryo; Cytoplasmic and Nuclear/genetics/*physiology; Mammalian; Retinoic Acid/genetics/physiology
Hydrophobic bile acids strongly repressed transcription of the human cholesterol 7alpha-hydroxylase gene (CYP7A1) in the bile acid biosynthetic pathway in the liver. 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 alpha-fetoprotein transcription factor (FTF) and small heterodimer partner (SHP) mRNA expression in HepG2 cells. Feeding of rats with chenodeoxycholic acid repressed CYP7A1, induced FTF, but had no effect on SHP mRNA expression in the liver. FTF 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 FTF 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; Owsley E; Yang Y; Stroup D; Chiang J Y
Journal of lipid research
2001
2001-09
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Identification of a bile acid response element in the cholesterol 7 alpha-hydroxylase gene CYP7A.
Animals; Base Sequence; Bile Acids and Salts/*pharmacology; Cholesterol 7-alpha-Hydroxylase/*genetics; Cultured; DNA-Binding Proteins/metabolism; Feedback; Genes; Genes/*drug effects; Luciferases/genetics; Rats; Reporter; Tumor Cells
The transcriptional activity of the cholesterol 7 alpha-hydroxylase gene CYP7A is repressed by bile acids. Taurine conjugates of chenodeoxycholate and deoxycholate, but not cholate and ursodeoxycholate, inhibited the CYP7A promoter/luciferase reporter activity in transient transfection assays in Hep G2 cells. A region from nucleotide (nt) -74 to -55 was found to mediate bile acid response. However, deletion of this bile acid response element (BARE-I) enhanced reporter activity but did not eliminate the bile acid response. This is due to the presence of another BARE-II located in a conserved region between nt -149 and -128. Deletion or mutations of these sequences reduced promoter activity and abolished bile acid repression. This BARE-II shares an identical AGTTCAAG core sequence with BARE-I. Electrophoretic mobility shift assays of BARE-I and BARE-II probes using Hep G2 nuclear extract and the partially purified binding activity of nt -65/-54 DNA-affinity column revealed that the same or a similar nuclear protein might bind to both BAREs. BARE-II is the major BARE involved in the transcriptional repression of the CYP7A gene by hydrophobic bile acids.
Stroup D; Crestani M; Chiang J Y
The American journal of physiology
1997
1997-08
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1152/ajpgi.1997.273.2.G508" target="_blank" rel="noreferrer noopener">10.1152/ajpgi.1997.273.2.G508</a>