[Show abstract][Hide abstract] ABSTRACT: Microsomal epoxide hydrolase (mEH) is a bifunctional protein that plays a central role in the metabolism of numerous xenobiotics as well as mediating the sodium-dependent transport of bile acids into hepatocytes. These compounds are involved in cholesterol homeostasis, lipid digestion, excretion of xenobiotics and the regulation of several nuclear receptors and signaling transduction pathways. Previous studies have demonstrated the critical role of GATA-4, a C/EBPα-NF/Y complex and an HNF-4α/CAR/RXR/PSF complex in the transcriptional regulation of the mEH gene (EPHX1). Studies also identified heterozygous mutations in human EPHX1 that resulted in a 95% decrease in mEH expression levels which was associated with a decrease in bile acid transport and severe hypercholanemia. In the present investigation we demonstrate that EPHX1 transcription is significantly inhibited by two heterozygous mutations observed in the Old Order Amish population that present numerous hypercholanemic subjects in the absence of liver damage suggesting a defect in bile acid transport into the hepatocyte. The identity of the regulatory proteins binding to these sites, established using biotinylated oligonucleotides in conjunction with mass spectrometry was shown to be poly(ADP-ribose)polymerase-1 (PARP-1) bound to the EPHX1 proximal promoter and a linker histone complex, H1.2/Aly, bound to a regulatory intron 1 site. These sites exhibited 71% homology and may represent potential nucleosome positioning domains. The high frequency of the H1.2 site polymorphism in the Amish population results in a potential genetic predisposition to hypercholanemia and in conjunction with our previous studies, further supports the critical role of mEH in mediating bile acid transport into hepatocytes.
[Show abstract][Hide abstract] ABSTRACT: Microsomal epoxide hydrolase (mEH) is a bifunctional protein that plays a central role in the metabolism of numerous xenobiotics as well as mediating the sodium-dependent transport of bile acids into hepatocytes where they are involved in cholesterol excretion and metabolism, lipid digestion and regulating numerous signaling pathways. Previous studies have demonstrated the critical role of GATA-4 and a C/EBPα-NF/Y complex in the regulation of the mEH gene (EPHX1). In this study we show that HNF-4α and CAR/RXR also bind to the proximal promoter region and regulate EPHX1 expression. Bile acids, which inhibit the expression of HNF-4α also decrease the expression of EPHX1. Studies also established that the binding of HNF-4α was essential for the activation of EPHX1 activity by CAR suggesting the formation of a complex between these adjacent factors. The nature of this regulatory complex was further explored using a biotinylated oligonucleotide of this region in conjunction with BioMag beads and mass spectrometric analysis which demonstrated the presence of an additional inhibitory factor (PSF), confirmed by co-immunoprecipitation and ChIP analyses, which interacted with DNA-bound CAR/RXR/HNF-4α forming a 4-component regulatory complex.
No preview · Article · May 2013 · Biochimica et Biophysica Acta
[Show abstract][Hide abstract] ABSTRACT: Amelogenin is the major protein component of the forming enamel matrix. In situ hybridization revealed a periodicity for amelogenin mRNA hybridization signals ranging from low to high transcript abundance on serial sections of developing mouse teeth. This in vivo observation led us to examine the amelogenin promoter for the activity of transcription factor(s) that account for this expression aspect of the regulation for the amelogenin gene. We have previously shown that CCAAT/enhancer-binding protein alpha (C/EBPalpha) is a potent transactivator of the mouse X-chromosomal amelogenin gene acting at the C/EBPalpha cis-element located in the -70/+52 minimal promoter. The minimal promoter contains a reversed CCAAT box (-58/-54) that is four base pairs downstream from the C/EBPalpha binding site. Similar to the C/EBPalpha binding site, the integrity of the reversed CCAAT box is also required for maintaining the activity of the basal promoter. We therefore focused on transcription factors that interact with the reversed CCAAT box. Using electrophoretic mobility shift assays we demonstrated that NF-Y was directly bound to this reversed CCAAT site. Co-transfection of C/EBPalpha and NF-Y synergistically increased the promoter activity. In contrast, increased expression of NF-Y alone had only marginal effects on the promoter. A dominant-negative DNA binding-deficient NF-Y mutant (NF-YAm29) dramatically decreased the promoter activity both in the absence or presence of exogenous expression of C/EBPalpha. We identified protein-protein interactions between C/EBPalpha and NF-Y by a co-immunoprecipitation analysis. These results suggest that C/EBPalpha and NF-Y synergistically activate the mouse amelogenin gene and can contribute to its physiological regulation during amelogenesis.
[Show abstract][Hide abstract] ABSTRACT: Microsomal epoxide hydrolase (mEH) plays a central role in xenobiotic metabolism as well as mediating the sodium-dependent uptake of bile acids into the liver, where these compounds regulate numerous biological processes such as cholesterol metabolism and hepatocyte signaling pathways. Little is known, however, about the factors that control the constitutive and inducible expression of the mEH gene (EPHX1) that is altered during development and in response to numerous xenobiotics. In previous studies we have established that GATA-4 binding to the EPHX1 core promoter is critical for EPHX1 expression. The -80/+25 bp core promoter also contained a reversed CCAAT box (-5/-1 bp), integrity of which was required for maximal basal EPHX1 transcription in HepG2 cells. Transient transfection of CCAAT/enhancer-binding protein α (C/EBPα) substantially stimulated EPHX1 promoter activity. Electrophoretic mobility shift assays, however, revealed that nuclear factor Y (NF-Y), but not C/EBPα, directly bound to this site although increased expression of NF-Y had no effect on EPHX1 promoter activity. These results suggested that C/EBPα activated EPHX1 expression through its interaction with NF-Y bound to the CCAAT box. The existence of a C/EBPα[NF-Y] complex was supported by electrophoretic mobility shift assays using antibodies against NF-Y and C/EBPα as well as by the ability of a dominant-negative NF-Y expression vector to inhibit promoter activity. The interaction between these transcription factors was established by co-immunoprecipitation analysis and glutathione S-transferase pull-down assays, whereas the association of the two factors and the interaction of NF-Y with the CCAAT box in vivo was confirmed by chromatin immunoprecipitation assays. C/EBPα-dependent EPHX1 activation was also supported by reconstitution studies in HeLa cells that lack this protein. These results establish that EPHX1 expression is regulated by C/EBPα interacting with DNA-bound NF-Y.
Preview · Article · Aug 2004 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Microsomal epoxide hydrolase (mEH) is a bifunctional protein that plays a crucial role in the metabolism of numerous xenobiotics as well as in mediating the hepatic sodium-dependent uptake of bile acids that are involved in numerous physiological processes including the regulation of cholesterol metabolism. The transcription factors and nuclear receptors that control the constitutive and inducible expression of the mEH gene (EPHX1), however, have not been described. To characterize these factors, a series of 5'-deletion constructs have been transfected into human liver-derived HepG2 cells as well as non-hepatic HeLa cells. Promoter activity analysis indicated the presence of a positive regulatory element in the -80/-70 bp region. Sequence analysis revealed a putative GATA site at -79/-74 bp as well as an additional site at -31/-26 bp. Electrophoretic mobility shift assays with an anti-GATA-4 antibody confirmed that GATA-4 bound to these two sites with a dissociation constant of 1.56 nM (-79 site) and 0.65 nM (-31 site). Coexpression of GATA-4 stimulated EPHX1 promoter activity up to 7.5-fold in a dose-dependent manner. Endogenous EPHX1 message in HepG2 cells was also significantly increased by overexpression of GATA-4. Mutating the -79 element resulted in a 65% loss of promoter activity, while mutating the -31 element had no effect on basal activity but greatly reduced the response to additional GATA-4. In HeLa cells, which do not express GATA-4, EPHX1 activity was negligible; however, activity could be reconstituted by the addition of exogenous GATA-4. These results demonstrate that GATA-4 plays a critical role in regulating EPHX1 expression.
No preview · Article · Mar 2004 · Biochimica et Biophysica Acta
[Show abstract][Hide abstract] ABSTRACT: The bifunctional hepatic protein, microsomal epoxide hydrolase (mEH), plays a central role in the metabolism of many xenobiotics as well as mediating the Na(+)-dependent uptake of bile acids in parallel with the Na(+)-taurocholate co-transporting protein (ntcp). Previous studies have established that mEH is expressed in the endoplasmic reticulum with two topological orientations, where the type II form is targeted to the plasma membrane. In this report the topology and transport properties of mEH as a function of plasma membrane expression in cultured hepatocytes, transfected Madin-Darby canine kidney cells expressing mEH (MDCK[mEH]), and the human hepatoma cell line, HepG2, were studied using confocal fluorescence microscopy and substrate uptake measurements. Analysis of mEH localization with an anti-mEH monoclonal antibody demonstrated the expression of one topological form on the plasma membrane of hepatocytes and MDCK[mEH] cells where both systems exhibited Na(+)-dependent bile acid uptake. In contrast, Na(+)-dependent bile acid transport in HepG2 cells and hepatocytes in culture (72 h) was substantially reduced as was the expression of ntcp. Although the total mEH level was undiminished, the decrease of bile acid transport was associated with the loss of mEH surface expression possibly resulting from an alteration in mEH endoplasmic reticulum topology and/or the plasma membrane protein targeting system in these de-differentiated cells.
No preview · Article · Nov 2003 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Microsomal epoxide hydrolase (mEH) is a bifunctional protein that plays a central role in carcinogen metabolism and is also able to mediate the sodium-dependent uptake of bile acids into hepatocytes. Studies have identified a subject (S-1) with extremely elevated serum bile salt levels in the absence of observable hepatocellular injury, suggesting a defect in bile acid uptake. In this individual, mEH protein and mEH mRNA levels were reduced by approximately 95% and 85%, respectively, whereas the expression and amino acid sequence of another bile acid transport protein (NTCP) was unaffected. Sequence analysis of the mEH gene (EPHX1) revealed a point mutation at an upstream HNF-3 site (allele I) and in intron 1 (allele II), which resulted in a significant decrease in EPHX1 promoter activity in transient transfection assays. Gel shift assays using a radiolabeled oligonucleotide from each region resulted in specific transcription factor binding patterns, which were altered in the presence of the mutation. These studies demonstrate that the expression of mEH is greatly reduced in a patient with hypercholanemia, suggesting that mEH participates in sodium-dependent bile acid uptake in human liver where its absence may contribute to the etiology of this disease.
Full-text · Article · Aug 2003 · Biochimica et Biophysica Acta
[Show abstract][Hide abstract] ABSTRACT: Microsomal epoxide hydrolase (mEH) is a bifunctional membrane protein that plays a central role in the metabolism of xenobiotics
and in the hepatocyte uptake of bile acids. Numerous studies have established that this protein is expressed both in the endoplasmic
reticulum and at the sinusoidal plasma membrane. Preliminary evidence has suggested that mEH is expressed in the endoplasmic
reticulum (ER) membrane with two distinct topological orientations. To further characterize the membrane topology and targeting
of this protein, an N-glycosylation site was engineered into mEH to serve as a topological probe for the elucidation of the cellular location of
mEH domains. The cDNAs for mEH and this mEH derivative (mEHg) were then expressed in vitro and in COS-7 cells. Analysis of total expressed protein in these systems indicated that mEHg was largely unglycosylated,
suggesting that expression in the ER was primarily of a type I orientation (Ccyt/Nexo). However, analysis, by biotin/avidin
labeling procedures, of mEHg expressed at the surface of transfected COS-7 cells, showed it to be fully glycosylated, indicating
that the topological form targeted to this site originally had a type II orientation (Cexo/Ncyt) in the ER. The surface expression
of mEH was also confirmed by confocal fluorescence scanning microscopy. The sensitivity of mEH topology to the charge at the
N-terminal domain was demonstrated by altering the net charge over a range of 0 to +3. The introduction of one positive charge
led to a significant inversion in mEH topology based on glycosylation site analysis. A truncated form of mEH lacking the N-terminal
hydrophobic transmembrane domain was also detected on the extracellular surface of transfected COS-7 cells, demonstrating
the existence of at least one additional transmembrane segment. These results suggest that mEH may be integrated into the
membrane with multiple transmembrane domains and is inserted into the ER membrane with two topological orientations, one of
which is targeted to the plasma membrane where it mediates bile acid transport.
No preview · Article · Sep 1999 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The 5' flanking sequence of the human monoamine oxidase A (MAO A) gene consists of an extensive repeat structure. Two 90-bp repeats (I and II) were found in the core promoter (the 0.24-kb PvuII/DraII fragment), each containing two Sp1 binding sites. An additional six repeats were found, five of which (III-VII) were upstream and one (-I) downstream of the core promoter. Using transient transfection assay with a human growth hormone reporter gene, we found that the upstream repeating units III-VII (in a 0.78-kb BamHI/DraII fragment) down-regulate core promoter activity to 13 +/- 10% in a human glioma cell line (1242 MG) and 2 +/- 1% in a cervical carcinoma cell line (HeLa), respectively. The 0.24-kb core promoter activity was taken as 100%. Addition of the initiator (Inr)-like sequence to this 0.78-kb fragment (0.82-kb BamHI/-17 fragment) still showed decreased promoter activity (10 +/- 9% in 1242 MG cells and 8 +/- 1% in HeLa cells). Thus, the upstream sequence down-regulates promoter activity with or without the Inr-like sequence. When the Inr-like sequence was added to the core promoter (0.28-kb PvuII/-17 fragment), the promoter activity decreases significantly in both 1242 MG (55 +/- 6%) and HeLa (60 +/- 10%) cells. These results suggest that although the Inr-like sequence is present in the human MAO A promoter, it acts as a negative cis element instead of a transcription initiator.
Full-text · Article · Nov 1997 · Journal of Neurochemistry
[Show abstract][Hide abstract] ABSTRACT: This chapter discusses the expression of the human monoamine oxidase (MAO) A gene controlled by transcription factor Sp1. There are four potential Spl binding sites in the MAO A promoter. However, it is not clear whether Spl is the major transcription factor binding to the MAO A promoter because a number of other transcription factors also bind to Spl sites. To see whether there are additional transcription factor binding sites, a DNasel footprinting experiment was performed in a study discussed in the chapter. DNasel digests the DNA fragment at random positions. In the presence of nuclear extract that contains various transcription factors, the DNA sequences bound by proteins are protected from DNasel digestion and appear as “bleached” areas in the DNA ladder. The 0.24 kb MA0 A promoter fragment radiolabeled at one end was partially digested with DNasel in the presence and absence of nuclear proteins from SHSY-5Y (human neuroblastoma) cells expressing MAO A. It was found in the study that only the four Spl binding sites are protected suggesting that these four sites are the major DNA sequences interacting with transcription factors. To see whether the transcription factor interacting with the four sites is Spl, a gel retardation experiments was performed in which a radio-labeled A0.24 fragment was incubated with SHSY-5Y nuclear proteins.
No preview · Article · Dec 1995 · Progress in brain research
[Show abstract][Hide abstract] ABSTRACT: The regulation of 5-HT2A receptor (5-HT2AR) expression has been implicated in a variety of pathological processes. Previous data addressing the regulation of this receptor are extremely complicated and controversial. In order to understand the mechanisms of regulation of this receptor, we have identified the promoter region of the human 5-HT2AR gene. Anchored PCR has mapped a cluster of transcription initiation sites at nucleotides -1157, -1137, -1127 (numbered sequentially as sites 1, 2 and 3). An additional initiation site (site 4) was detected at -496, 631 bp downstream of site 3. Promoter activity was defined by transfection studies. Several 5′ flanking fragments linked to the human growth hormone reporter gene were transfected into two human cell lines, SHSY-5Y (neuroblastoma) and HeLa (cervic carcinoma) which express 5-HT2AR. A 0.74 kb HaeIII/PvuII fragment, which encompasses the initiation sites 1 to 3 and 5′ of the downstream initiation site 4, exhibited significant promoter activity in both cell lines. Inclusion of additional sequences upstream (the 1.6 kb PvuII/PvuII fragment) had little effect on the promoter activity, but the extension of the 0.74 kb fragment downstream to include a 0.45 kb PvuII/SmaI fragment drastically decreased the promoter activity. These results suggest that the promoter activity for human 5-HT2AR gene resides in the 0.74 kb HaeIII/PvuII fragment and the 0.45 kb PvuII/SmaI fragment may contain a silencer for the gene expression.
No preview · Article · Dec 1995 · Behavioural Brain Research