Qin-shi Zhu

University of Southern California, Los Ángeles, California, United States

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Publications (10)37.71 Total impact

  • [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.
    Biochimica et Biophysica Acta 05/2013; 1829(10). DOI:10.1016/j.bbagrm.2013.05.003 · 4.66 Impact Factor
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    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.
    Journal of Biological Chemistry 07/2006; 281(23):16090-8. DOI:10.1074/jbc.M510514200 · 4.60 Impact Factor
  • Qin-Shi Zhu, Bin Qian, Daniel Levy
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    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 alpha (C/EBPalpha) substantially stimulated EPHX1 promoter activity. Electrophoretic mobility shift assays, however, revealed that nuclear factor Y (NF-Y), but not C/EBPalpha, directly bound to this site although increased expression of NF-Y had no effect on EPHX1 promoter activity. These results suggested that C/EBPalpha activated EPHX1 expression through its interaction with NF-Y bound to the CCAAT box. The existence of a C/EBPalpha[NF-Y] complex was supported by electrophoretic mobility shift assays using antibodies against NF-Y and C/EBPalpha 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/EBPalpha-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/EBPalpha interacting with DNA-bound NF-Y.
    Journal of Biological Chemistry 08/2004; 279(29):29902-10. DOI:10.1074/jbc.M400438200 · 4.60 Impact Factor
  • Qin-shi Zhu, Bin Qian, Daniel Levy
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    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.
    Biochimica et Biophysica Acta 03/2004; 1676(3):251-60. DOI:10.1016/j.bbaexp.2004.01.002 · 4.66 Impact Factor
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    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.
    Biochemical and Biophysical Research Communications 11/2003; 309(4):804-9. DOI:10.1016/j.bbrc.2003.08.074 · 2.28 Impact Factor
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    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.
    Biochimica et Biophysica Acta 08/2003; 1638(3):208-16. DOI:10.1016/S0925-4439(03)00085-1 · 4.66 Impact Factor
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    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.
    Journal of Biological Chemistry 09/1999; 274(39):27898-27904. · 4.60 Impact Factor
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    Qin-shi Zhu, Jean Chen Shih
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    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.
    Journal of Neurochemistry 11/1997; 69(4):1368-73. DOI:10.1046/j.1471-4159.1997.69041368.x · 4.24 Impact Factor
  • Jean Chen Shih, Qin-shi Zhu, Kevin Chen
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    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.
    Behavioural Brain Research 12/1995; 73(1):59-62. DOI:10.1016/0166-4328(96)00070-8 · 3.39 Impact Factor
  • Jean C. Shih, Qin-shi Zhu, Kevin Chen