Studies on the transcriptional regulation of sterol 12alpha-hydroxylase (CYP8B1)
Author: Yang, Yizeng
Date: 2002-05-28
Location: Huddinge Universitetssjukhus M63
Time: 10.00
Department: Institutionen för medicinsk laboratorievetenskap och teknik / Department of Laboratory Sciences and Technology
Abstract
The conversion of cholesterol into bile acids is mainly via two pathways,
which are initiated by CYP7A1 and CYP27A1 respectedly. CYP8B1, a
microsomal P450 cytochrome, hydroxylates of the steroid nucleus at the
C-12alpha position to form 7alpha, 12alpha-dihydroxy-4-cholesten-3-one, a
facultative precursor of CA. The activity of the enzyme determines the
ratio between CA and CDCA. The enzyme activity is affected by bile acids,
cholesterol, hormones and fasting. In the present studies, we have
focused on the structure-function relationship of the rat gene to explore
the mechanisms of regulation.
The cDNA for rat CYP8B1 coded for a protein of 499 amino acid residues.
The gene, like the human and mouse gene, also lacked introns. The
promoter region contains several putative transcriptional response
elements including DR1, c-jun, NF-1, Sp1 and repeated SRE-1 motifs.
Thyroidectomy in rats caused a four-fold increase in CYP8B1 mRNA in the
liver together with a 2,2-fold increase of enzyme activity. The treatment
of intact animals with L-thyroxine caused a 60% reduction of the enzyme
activity and a 50% reduction in CYP8B1 mRNA. No putative DR4 or thyroid
hormone response elements could be recognized in the promoter fragment.
Previous studies in rats showed that CYP8B1 activity and mRNA levels were
increased by fasting and clofibrate. In present studies, fasting for 24
hours or administration of WY-14,643 did not change the CYP8B1 activity
and mRNA levels in PPAR-alpha null mice, whereas a significant increase
was recorded in wild-type animals. An relative increase of CA was found
in the bile. In vitro, PPAR-alpha was found to directly bind to a DR1
motif in the promoter. Co-transfection with PPAR-alpha expression
plasmids induced a 2,5-fold increase of the activity of the CYP8B1
promoter in a ligand-dependent manner in Hep G2 cells.
Similar to CYP7A1, CYP8B1 is subjected to a negative feed-back regulation
by bile acids. Feeding rats with chenodeoxycholic acid caused a 40%
decrease of the liver CYP8B1 mRNA levels, while cholestyramine increased
the mRNA 120%. It was associated with an increased mRNA levels for FTF
and decreased for HNF4-alpha. A bile acid response element (BARE), DR1
motif overlapped with two M binding sites, was identified. FTF strongly
repressed the CYP8B1 promoter activity probably by a competitive binding
to BARE with HNF4-alpha.
In contrast to the up-regulation of CYP7A1, cholesterol feeding of
rodents decreases the activity of CYP8B1. Treatment with
cholesterol/25-hydroxycholesterol inhibited the activity of the rat
CYP8B1 promoter in a dose-dependent manner. Overexpression of SREBPl a
and l c stimulated the activity of promoter from rat, mouse and man,
while SREBP2 did not have any effects. SREBP1 a and SREBP1 c directly
bound to SRE and E box motifs in the rat promoter. Cholesterol feeding
also decreased the mRNA levels for SREBP1, but not for SREBP2 in rat
liver.
In summary, the HNF4-alpha, FTF and PPAR-alpha mediate the regulation of
CYP8B1 by bile acids and fatty acids through BARE in the gene promoter.
The coordinative effect between HNF4-alpha and FTF seems to be crucial
for the expression level of CYP8B. PPAR-alpha mediates the up-regulation
of CYP8B1 during starvation and fibrate administration. SREBP1 promotes
the transcription of CYP8B1 gene by interaction with SRE and E box
motifs, thereby constituting a new regulatory link between the
cholesterol and bile acid metabolism. The CYP8B1 gene is also subjected
to down-regulation by thyroid hormone, but responsive elements have not
yet been identified in the promoter.
List of papers:
I. Andersson U, Yang YZ, Bjorkhem I, Einarsson C, Eggertsen G, Gafvels M (1999). "Thyroid hormone suppresses hepatic sterol 12alpha-hydroxylase (CYP8B1) activity and messenger ribonucleic acid in rat liver: failure to define known thyroid hormone response elements in the gene. " Biochim Biophys Acta 1438(2): 167-74
Pubmed
II. Hunt MC, Yang YZ, Eggertsen G, Carneheim CM, Gafvels M, Einarsson C, Alexson SE (2000). "The peroxisome proliferator-activated receptor alpha (PPARalpha) regulates bile acid biosynthesis. " J Biol Chem 275(37): 28947-53
Pubmed
III. Yang Y, Zhang M, Eggertsen G, Chiang JYL (2002). "On the mechanism of bile acid inhibition of rat sterol 12alpha-hydroxylase gene (CYP8B1) transcription: roles of alpha-fetoprotein transcription factor and hepatocyte nuclear factor 4alpha." Biochim Biophys Acta (In Print)
IV. Yang Y, Gafvels M, Andersson U, Einarsson C, Bjorkhem I, Chiang JYL, Eggertsen G (2002). "Mechanisms of regulation of sterol 12alpha-hydroxylase (CYP8B1) by cholesterol. Species dependent variations due to varying numbers of SRE and E box motifs." (Submitted)
I. Andersson U, Yang YZ, Bjorkhem I, Einarsson C, Eggertsen G, Gafvels M (1999). "Thyroid hormone suppresses hepatic sterol 12alpha-hydroxylase (CYP8B1) activity and messenger ribonucleic acid in rat liver: failure to define known thyroid hormone response elements in the gene. " Biochim Biophys Acta 1438(2): 167-74
Pubmed
II. Hunt MC, Yang YZ, Eggertsen G, Carneheim CM, Gafvels M, Einarsson C, Alexson SE (2000). "The peroxisome proliferator-activated receptor alpha (PPARalpha) regulates bile acid biosynthesis. " J Biol Chem 275(37): 28947-53
Pubmed
III. Yang Y, Zhang M, Eggertsen G, Chiang JYL (2002). "On the mechanism of bile acid inhibition of rat sterol 12alpha-hydroxylase gene (CYP8B1) transcription: roles of alpha-fetoprotein transcription factor and hepatocyte nuclear factor 4alpha." Biochim Biophys Acta (In Print)
IV. Yang Y, Gafvels M, Andersson U, Einarsson C, Bjorkhem I, Chiang JYL, Eggertsen G (2002). "Mechanisms of regulation of sterol 12alpha-hydroxylase (CYP8B1) by cholesterol. Species dependent variations due to varying numbers of SRE and E box motifs." (Submitted)
Issue date: 2002-05-07
Publication year: 2002
ISBN: 91-628-5275-2
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