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1.
Mediators Inflamm ; 2016: 7945848, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27293321

RESUMO

Targeting the endothelial adhesion molecules that control leukocyte trafficking into a tissue has been explored as a biological therapy for inflammatory diseases. However, these molecules also participate in leukocyte migration for immune surveillance, and inhibiting the physiological level of an adhesion molecule might promote infection or malignancy. We explored the concept of targeting endothelial adhesion molecule transcription during inflammation in a human system. Intercellular adhesion molecule 1 (ICAM-1) mediates leukocyte migration across the retinal endothelium in noninfectious posterior uveitis. We observed an increase in the transcription factor, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NF-κB1), in parallel with ICAM-1, in human retinal endothelial cells treated with tumor necrosis factor-alpha (TNF-α), and identified putative binding sites for NF-κB1 within the ICAM-1 regulatory region. We targeted induced NF-κB1 expression in endothelial cells with small interfering (si)RNA. Knockdown of NF-κB1 significantly decreased cell surface expression of ICAM-1 protein induced by TNF-α but did not reduce constitutive ICAM-1 expression. Consistently, NF-κB1 knockdown significantly reduced leukocyte binding to cell monolayers in the presence of TNF-α but did not impact baseline binding. Findings of this proof-of-concept study indicate that induced transcription of endothelial adhesion molecules might be targeted therapeutically for inflammatory disease in humans.


Assuntos
Inflamação/genética , Molécula 1 de Adesão Intercelular/genética , Retina/metabolismo , Adesão Celular , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Leucócitos/citologia , Leucócitos/metabolismo , Interferência de RNA/fisiologia , Retina/citologia , Retina/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/farmacologia
2.
Mol Pharmacol ; 78(4): 714-22, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20628005

RESUMO

UDP glucuronosyltransferase 2B17 is present in the prostate, where it catalyzes the addition of glucuronic acid to testosterone and dihydrotestosterone and their metabolites androsterone and androstane-3α,17ß-diol. Hence, changes in UGT2B17 gene expression may affect the capacity of the prostate to inactivate and eliminate male sex hormones. In this work, we identify a prevalent polymorphism, -155G/A, in the proximal promoter of the UGT2B17 gene. This polymorphism modulates UGT2B17 promoter activity, because luciferase-gene reporter constructs containing the -155A allele were 13-fold more active than those containing the -155G allele in prostate cancer LNCaP cells. The -155G/A polymorphism is contained within a putative binding site for the transcription factor Forkhead Box A1 (FOXA1). Using gene reporter, electromobility shift, and chromatin immunoprecipitation analyses, we show that FOXA1 binds to this site and stimulates the UGT2B17 promoter. Furthermore, down-regulation of FOXA1 in LNCaP cells substantially reduces UGT2B17 mRNA levels. The binding of FOXA1 and subsequent stimulation of the UGT2B17 promoter is greatly reduced in the presence of the -155G allele compared with the -155A allele. Consonant with its capacity to be stimulated by FOXA1, the UGT2B17 -155A allele, compared with the -155G allele, is associated with higher levels of circulating androstane-3α,17ß-diol glucuronide. Although the initial phases of prostate cancer are androgen-dependent and UGT2B17 inactivates androgens, there was no association of the UGT2B17 -155G/A polymorphism with prostate cancer risk. In summary, this work identifies FOXA1 as an important regulator of UGT2B17 expression in prostate cancer LNCaP cells and identifies a polymorphism that alters this regulation.


Assuntos
Androstano-3,17-diol/análogos & derivados , Glucuronosiltransferase/genética , Fator 3-alfa Nuclear de Hepatócito/genética , Polimorfismo Genético/fisiologia , Regiões Promotoras Genéticas/fisiologia , Androstano-3,17-diol/sangue , Sítios de Ligação/fisiologia , Linhagem Celular Tumoral , Glucuronosiltransferase/metabolismo , Fator 3-alfa Nuclear de Hepatócito/sangue , Humanos , Masculino , Antígenos de Histocompatibilidade Menor , Neoplasias da Próstata/sangue , Neoplasias da Próstata/genética
3.
Drug Metab Rev ; 42(1): 99-109, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20070244

RESUMO

Elucidation of the mechanisms regulating UGT genes is of prime importance if the adverse effects of interactions between drugs primarily eliminated by glucuronidation are to be minimized, and if UGT expression is to be manipulated for therapeutic effect. The factors controlling UGT gene expression in the liver include the liver-enriched transcription factors, HNF-1alpha and HNF-4alpha, several members of the nuclear-receptor family (CAR, PXR, FXR, LXR, and PPAR), the arylhydrocarbon receptor, and transcription factors involved in stress responses (Nrf2, Maf). HNF-1alpha, in concert with the intestine-specific transcription factor, Cdx2, and Sp1 regulate UGT gene expression in the gastrointestinal tract, whereas the genes for the major androgen-glucuronidating enzymes, UGT2B15 and UGT2B17, are upregulated by estrogens in breast cell lines and downregulated by androgens in prostate-derived cells. Despite this knowledge, the complex interactions between these transcription factors and their coregulators has not been determined, and the mechanisms regulating UGT gene expression in organs and tissues, other than the liver, gastrointestinal tract, breast, and prostate, remain to be elucidated.


Assuntos
Fatores Ativadores da Transcrição/fisiologia , Regulação Enzimológica da Expressão Gênica , Glucuronosiltransferase/metabolismo , Regiões Promotoras Genéticas/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Regulação Neoplásica da Expressão Gênica , Glucuronosiltransferase/genética , Humanos , Mucosa Intestinal/metabolismo , Masculino , Proteínas Nucleares/fisiologia , Próstata/enzimologia
4.
Pharmacogenet Genomics ; 17(1): 25-36, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17264800

RESUMO

In humans, UDP-glucuronosyltransferase 1A9 is known to glucuronidate numerous lipophilic substances of pharmacological and toxicological importance. Although it has been established that individuals vary in their capacity to express this detoxification enzyme, little is known about the mechanisms that dictate the regulation of UGT1A9. In particular, it is not understood why, while the proximal regulatory regions of the UGT1A7-10 gene cluster are highly similar, UGT1A9 is the sole hepatic isoform of the four. Recent data have suggested that the human UGT1A9 promoter is controlled by hepatocyte nuclear factor 4alpha. In this work, we confirm that the human UGT1A9 promoter can indeed be upregulated by human hepatocyte nuclear factor 4alpha in vitro. Our results, however, show that the previously-reported hepatocyte nuclear factor 4alpha-binding site only plays a minor role in this response. Instead, upregulation was found to require a more proximal response element, which was not preserved in the UGT1A7, UGT1A8 or UGT1A10 promoters. Furthermore, hepatocyte nuclear factor 4alpha-mediated transcription from the human UGT1A9 promoter was discovered to be entirely dependent on hepatocyte nuclear factor 1. We have established that two hepatocyte nuclear factor 1-binding elements are involved in this phenomenon, the more distal of which is unique to the UGT1A9 promoter. Interestingly, this second site had no significant role in hepatocyte nuclear factor 1alpha-mediated induction of the UGT1A9 promoter in vitro, yet was critical for upregulation by human hepatocyte nuclear factor 4alpha. The discovery of two unique and cooperative liver-enriched transcription factor binding sites in the UGT1A9 promoter is a significant step towards understanding the unique hepatic expression of UGT1A9 amongst the UGT1A7-10 gene cluster.


Assuntos
Glucuronosiltransferase/genética , Fator 1 Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Ativação Transcricional , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Glucuronosiltransferase/metabolismo , Humanos , Fígado/enzimologia , Fígado/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Alinhamento de Sequência , UDP-Glucuronosiltransferase 1A , Regulação para Cima
5.
Drug Metab Dispos ; 35(1): 116-20, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17050649

RESUMO

The UGT1A3-1A5 genes are a highly related UDP-glucuronosyltransferase (UGT) cluster exhibiting high levels of coding and regulatory region homology. However, the ensuing proteins have both differing substrate specificities and differing expression patterns. The expression profile of each enzyme also varies considerably from one individual to the next. Differences in UGT expression have been predicted to contribute to an individual's response to pharmaceuticals and to predisposition toward cancer in the event of carcinogen exposure. Therefore, it is desirable to elucidate the mechanisms that drive the transcription of UGT genes and identify the factors responsible for their variable expression. To this end, we have isolated the UGT1A3, UGT1A4, and UGT1A5 proximal promoters and begun to investigate the regulatory elements necessary for activity in vitro. We have established that the nucleotide sequence upstream of the UGT1A5 exon 1 is an ineffective promoter, correlating with the lack of substantial expression of this UGT in human tissues. In contrast, the UGT1A3 and UGT1A4 proximal promoters are both highly active in hepatic and colonic cell lines, with maximal activity being encoded by the proximal 500 base pairs. However, the UGT1A3 and UGT1A4 promoters exhibit low activity in the human embryonic kidney cell line HEK293, unless coexpressed with hepatocyte nuclear factor (HNF) 1alpha. Furthermore, mutation of the consensus-like HNF1-binding site in the UGT1A3 promoter abolishes promoter function in all cell types. This study suggests an important role for HNF1alpha in the transcriptional regulation of the human UGT1A3 and UGT1A4 genes.


Assuntos
Glucuronosiltransferase/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Regiões Promotoras Genéticas , Células CACO-2 , Glucuronosiltransferase/metabolismo , Humanos , Transcrição Gênica
6.
Pharmacogenet Genomics ; 16(7): 527-36, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16788384

RESUMO

The gastrointestinal tract, contains several UDP-glucuronosyltransferases (UGTs) of the UGT1A and UGT2B subfamilies. UGT2B7 is one particular enzyme expressed throughout the gastrointestinal tract that possesses broad substrate specificity towards orally administered drugs. Because the caudal-related homeodomain protein 2 (Cdx2) regulates many gastrointestinal properties, we sought to determine whether it could regulate the UGT2B7 promoter in the colon-derived cell line Caco-2. Levels of Cdx2 and UGT2B7 were measured in differentiated and non-differentiated Caco-2 cells by the quantitative polymerase chain reaction. The capacity of the UGT2B7 gene promoter to drive expression of the luciferase reporter gene was assessed by transfection into Caco-2 cells, with transcription factor expression plasmids. Mutation of putative transcription factor binding sites and electrophoretic mobility shift assays were used to define important regulatory regions of the UGT2B7 gene promoter. The levels of Cdx2 and UGT2B7 mRNAs were co-ordinately increased in differentiated Caco2 cells compared to non-differentiated cells. Cdx2 activates the UGT2B7 proximal promoter by binding to two adjacent sites. Promoter activation requires Cdx2 binding to both sites wherein these proteins interact to form a putative functional dimer. Dimerization was shown to be dependent on redox state using extracts depleted of dithiothreitol. In addition, Cdx2 was shown to cooperatively activate the UGT2B7 promoter in conjunction with hepatocyte nuclear factor 1alpha (HNF1alpha), a mechanism previously observed to regulate other intestine-specific genes. The present study is the first to define transcription factors involved in the control of intestinal UGT2B expression. The demonstration that Cdx2 and HNF1alpha are important regulators of UGT2B7 expression will aid in defining pathways for coordinate control of drug metabolism in the gastrointestinal tract.


Assuntos
Glucuronosiltransferase/genética , Fator 1-alfa Nuclear de Hepatócito/fisiologia , Proteínas de Homeodomínio/fisiologia , Biotransformação/genética , Biotransformação/fisiologia , Fator de Transcrição CDX2 , Células CACO-2 , Regulação da Expressão Gênica , Glucuronosiltransferase/biossíntese , Humanos , Mutação , Regiões Promotoras Genéticas , Fatores de Transcrição/genética
7.
J Pharmacol Exp Ther ; 315(3): 1143-9, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16120810

RESUMO

The human UDP-glucuronosyltransferase (UGT) subfamily 1A includes nine genes. The expression of all the UGT1A isoforms, apart from UGT1A5, has been reported previously. We have now detected a low basal level of UGT1A5 expression in cultured human hepatocytes, and treatment with rifampicin or 3-methylcholanthrene increased the level of UGT1A5 mRNA. Low-level UGT1A5 expression was also found in HepG2 and Caco-2 cells as well as human liver. Furthermore, UGT1A5 expression has been detected in various segments of the intestine from human donors, revealing high interindividual variability in its level and distribution along the intestine. Full-length UGT1A5 cDNA was isolated from Caco-2 cells that had been transfected with the pregnane X receptor and treated with rifampicin. Recombinant UGT1A5, expressed in baculovirus-infected insect cells, exhibited very low rates of 4-methylumbelliferone and scopoletin glucuronidation, whereas 1-hydroxypyrene was a much better substrate for it. UGT1A5 did not glucuronidate 4-aminobiphenyl, a good substrate for the highly homologous enzymes UGT1A4 and UGT1A3. However, replacing the first 110 amino acids of UGT1A5, a region that may be involved in substrate binding, with the counterpart segment from UGT1A4 did not increase the 4-aminobiphenyl glucuronidation activity. Collectively, this work demonstrates for the first time that the human UGT1A5 is expressed in several tissues, is inducible, and is catalytically active.


Assuntos
Carcinoma Hepatocelular/enzimologia , Expressão Gênica , Glucuronosiltransferase/química , Glucuronosiltransferase/metabolismo , Hepatócitos/enzimologia , Neoplasias Hepáticas/enzimologia , Adulto , Idoso , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Células CACO-2 , Carcinoma Hepatocelular/patologia , Catálise , Linhagem Celular Tumoral , Células Cultivadas , DNA Complementar , Glucuronosiltransferase/análise , Glucuronosiltransferase/antagonistas & inibidores , Glucuronosiltransferase/genética , Hepatócitos/efeitos dos fármacos , Humanos , Insetos/citologia , Insetos/virologia , Mucosa Intestinal/enzimologia , Isoenzimas/análise , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Metilcolantreno/farmacologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rifampina/farmacologia , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
8.
Methods Enzymol ; 400: 22-46, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16399341

RESUMO

The hepatocyte nuclear factor 1 (HNF1) transcription factor family is composed of two closely related homeodomain proteins with similar but distinct expression profiles. Homodimers and heterodimers of these transcription factors, HNF1alpha and HNF1beta, increase transcription from target genes through direct physical interaction with one or more elements of sufficient similarity to a 13 nucleotide-inverted dyad consensus-binding sequence. Potential HNF1-binding sites have been found in the proximal upstream regulatory regions of most known human UDP-glucuronosyltransferase (UGT) genes. As the liver and gastrointestinal tract are both important sites of glucuronidation and express significant levels of one or both HNF1 proteins, it is thought that these homeoproteins may play a role in transcriptional regulation of UGTs. This chapter explores the current evidence that HNF1 transcription factors are explicitly involved in the transcription of mammalian UGT genes. Most data supporting this hypothesis come from in vitro reporter assays, site-directed mutagenesis, and electrophoretic mobility-shift assays, for which methods are detailed. However, as in vitro functionality of transcription factors does not necessarily imply significance in vivo, some of the limitations of these techniques are also examined. In addition, available in vivo data are discussed, with particular attention given to contributions made by HNF1alpha knockout mouse models and microarray studies of human tissue. Finally, possible scenarios in which HNF1-mediated regulation of UGT expression may be clinically relevant are suggested.


Assuntos
Glucuronosiltransferase/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 1-beta Nuclear de Hepatócito/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Western Blotting , Ensaio de Desvio de Mobilidade Eletroforética , Glucuronosiltransferase/biossíntese , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-beta Nuclear de Hepatócito/genética , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/fisiologia , Alinhamento de Sequência , Transcrição Gênica
9.
Toxicol Appl Pharmacol ; 199(3): 354-63, 2004 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-15364550

RESUMO

The UDP glucuronosyltransferases (UGT) of the gastrointestinal (GI) tract have a crucial role in protection against the toxic effects of lipophilic chemicals in the environment. UGTs such as UGT1A7, UGT1A8, and UGT1A10 are exclusively expressed in gastrointestinal tissues, each with a unique tissue distribution pattern that is subject to interindividual variation. The factors regulating this tissue-specific expression and that contribute to variability are beginning to be elucidated. Studies on the UGT1A7, 1A8, 1A9, and 1A10 gene promoters in Caco-2 cells, an in vitro model of enterocytes of the gastrointestinal tract, have identified the caudal homeodomain transcription factor, Cdx2, as an important regulator of the UGT1A8 and 1A10 gene proximal promoters. This transcription factor is found exclusively in the small intestine and colon: it is absent in the gastric epithelium and the esophagus. Cdx2 regulates the UGT1A8 and 1A10 promoters in cooperation with hepatocyte nuclear factor 1alpha (HNF1alpha). It is noteworthy that UGT1A7 is not expressed in gastrointestinal tissue distal to the gastric mucosa and does not contain a Cdx2 binding site in its proximal promoter. Transcription factors, including Sp1, which differentially bind to the initiator regions of the UGT1A8, 1A9, and 1A10 promoters, also contribute to the differences in expression of these UGTs in Caco-2 cells. The identification of important regulatory regions of UGT genes expressed in the gastrointestinal tract, and the transcription factors that bind to these regions, will aid in the elucidation of factors that contribute to interindividual differences in gastrointestinal UGT expression. In turn, this will lead to further understanding of interindividual variation in the capacity of the GI tract to metabolize lipophilic chemicals and to act as a barrier to dietary toxins and orally administered drugs.


Assuntos
Sistema Digestório/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Animais , Sistema Digestório/metabolismo , Enterócitos/enzimologia , Enterócitos/metabolismo , Glucuronídeos/metabolismo , Humanos
10.
Mol Pharmacol ; 65(4): 953-63, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15044625

RESUMO

The human UDP-glucuronosyltransferases (UGT) -1A8 and -1A10 are exclusively expressed in extrahepatic tissues and primarily in the gastrointestinal tract, whereas UGT1A9 is expressed mainly in the liver and kidneys. We have demonstrated previously that the UGT1A8 and UGT1A10 genes, in contrast to the UGT1A9 gene, are regulated via an initiator-like element in their proximal promoters. To determine the elements that contribute to the gastrointestinal expression of UGT1A8 and -1A10, we conducted deletion analysis of the UGT1A8, -1A9, and -1A10 promoters in the colon-derived cell line Caco2. DNA elements contributing significantly to UGT1A8, -1A9, and -1A10 promoter activity were found to reside primarily within 140 base pairs of the transcription start site. Within this region, putative binding sites for the intestine-specific transcription factor, caudal-related homeodomain protein 2 (Cdx2), and hepatocyte nuclear factor 1 (HNF1) were identified. Using gel shift and functional assays, HNF1alpha was demonstrated to bind to and activate the UGT1A8, -1A9, and -1A10 promoters. In contrast, Cdx2 bound to and activated the UGT1A8 and -1A10 promoters but could not activate the UGT1A9 promoter. A single base pair difference between the UGT1A8 and -1A10 promoters, three base pairs downstream of the consensus Cdx2 site, contributed to the observed difference in Cdx2 binding and Cdx2-mediated promoter activation of these two promoters. In addition, Cdx2 was shown to cooperate with HNF1alpha to synergistically activate the UGT1A8, -1A9, and -1A10 promoters. These studies provide insight into the mechanisms controlling the extrahepatic expression of the UGT1A8, -1A9, and -1A10 genes.


Assuntos
Regulação Enzimológica da Expressão Gênica , Glucuronosiltransferase/metabolismo , Proteínas de Homeodomínio/fisiologia , Proteínas Nucleares , Fatores de Transcrição/fisiologia , Fator de Transcrição CDX2 , Células CACO-2 , Proteínas de Ligação a DNA/fisiologia , Glucuronosiltransferase/genética , Fator 1 Nuclear de Hepatócito , Fator 1-alfa Nuclear de Hepatócito , Fator 1-beta Nuclear de Hepatócito , Proteínas de Homeodomínio/metabolismo , Humanos , Fígado/enzimologia , Regiões Promotoras Genéticas/fisiologia , Transativadores , UDP-Glucuronosiltransferase 1A
11.
Drug Metab Dispos ; 32(4): 413-23, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15039294

RESUMO

The glucuronidation kinetics of the prototypic substrates 4-methylumbelliferone (4MU) and 1-naphthol (1NP) by human UDP-glucuronosyltransferases (UGT) 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15, and 2B17 were investigated. Where activity was demonstrated, inhibitory effects of diclofenac, probenecid, and the solvents acetone, acetonitrile, dimethyl sulfoxide, ethanol, and methanol were characterized. All isoforms except UGT1A4 glucuronidated 4MU, whereas all but UGT 1A4, 2B15, and 2B17 metabolized 1NP. However, kinetic models varied with substrate (for the same isoform) and from isoform to isoform (with the same substrate). Hyperbolic (Michaelis-Menten), substrate inhibition, and sigmoidal kinetics were variably observed for both 4MU and 1NP glucuronidation by the various UGTs. K(m) or S(50) (sigmoidal kinetics) and V(max) values varied 525- (8-4204 microM) and 1386-fold, respectively, for 4MU glucuronidation, and 1360- (1.3-1768 microM) and 37-fold, respectively, for 1NP glucuronidation. The use of a two-site model proved useful for those reactions exhibiting non-Michaelis-Menten glucuronidation kinetics. The organic solvents generally had a relatively minor effect on UGT isoform activity. UGT 2B15 and 2B17 were most susceptible to the presence of solvent, although solvent-selective inhibition was occasionally observed with other isoforms. Diclofenac and probenecid inhibited all isoforms, precluding the use of these compounds for the reaction phenotyping of xenobiotic glucuronidation pathways in human tissues. Diclofenac and probenecid K(i) values, determined for selected isoforms, ranged from 11 to 52 microM and 96 to 2452 microM, respectively. Overall, the results emphasize the need for the careful design and interpretation of kinetic and inhibition studies with human UGTs.


Assuntos
Diclofenaco/farmacologia , Glucuronatos/farmacocinética , Glucuronosiltransferase/farmacologia , Himecromona/análogos & derivados , Himecromona/farmacocinética , Isoenzimas/farmacologia , Probenecid/farmacologia , Acetona/farmacologia , Animais , Western Blotting/métodos , Células CACO-2 , Dimetil Sulfóxido/farmacologia , Etanol/farmacologia , Glucuronatos/antagonistas & inibidores , Glucuronatos/metabolismo , Glucuronosiltransferase/antagonistas & inibidores , Glucuronosiltransferase/biossíntese , Humanos , Himecromona/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/biossíntese , Cinética , Metanol/farmacologia , Camundongos , Solventes/química , Solventes/farmacologia
12.
Drug Metab Dispos ; 32(3): 340-7, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14977869

RESUMO

The pregnane X receptor (PXR) has three known major transcript variants resulting from alternative splicing. The less well characterized variants T2 and T3 are identical to the well described variant T1 except for a 39-amino acid N-terminal extension in T2 and an internal 37-amino acid deletion in T3. We have developed reverse transcription-polymerase chain reaction (RT-PCR) methods to detect and quantify each human PXR (hPXR) in human liver and intestinal tissues and HepG2 and Caco-2 cell lines. All three isoforms were expressed in hepatic cells, whereas only T1 transcripts were found in Caco-2 cells. In general, most normal human liver and intestinal mucosa contained all three hPXR variants, but considerable interindividual variation in expression levels was found. The effect of each hPXR variant on expression of UDP-glucuronosyltransferase (UGT) UGT1A and UGT2B family isoforms was investigated in transiently transfected HepG2 and Caco-2 cells. As a family, UGT1A transcripts were up-regulated by T1 and T2 but not T3. Isoform-specific RT-PCR revealed that UGT1A1, 1A3, and 1A4 were the major isoforms induced in both cell lines. The levels of several UGT1A isoforms were also examined in human liver samples from a number of donors with characterized PXR expression. The data suggest that individual variation in PXR expression may account for differential expression of some UGT isoforms between subjects.


Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Glucuronosiltransferase/biossíntese , Glucuronosiltransferase/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Hidrocarboneto de Aril Hidroxilases/biossíntese , Hidrocarboneto de Aril Hidroxilases/genética , Células CACO-2 , Linhagem Celular , Clonagem Molecular , Citocromo P-450 CYP3A , Primers do DNA , Interações Medicamentosas , Indução Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Intestinos/enzimologia , Isoenzimas/biossíntese , Isoenzimas/genética , Fígado/enzimologia , Plasmídeos/genética , Receptor de Pregnano X , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
J Biol Chem ; 278(38): 36107-14, 2003 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-12847094

RESUMO

The human UDP-glucuronosyltransferases, UGT1A8, 1A9, and 1A10, are closely related in sequence and have a major role in the elimination of lipophilic chemicals by glucuronidation. UGT1A8 and 1A10 are expressed exclusively in the gastrointestinal tract, whereas UGT1A9 is expressed mainly in the liver and kidneys. To determine the factors contributing to the extrahepatic expression of these UDP-glucuronosyltransferases, we have cloned and characterized the promoters of the UGT1A8, 1A9, and 1A10 genes and studied their regulation in the colon cell line, Caco2. Their transcription start sites were mapped, and a functional overlapping Sp1/initiator-like site was identified which strongly contributed to UGT1A8 and 1A10 promoter activity. The high promoter activity of UGT1A8 and 1A10 correlated with the binding of nuclear proteins (complex B) to this region. Two-bp differences in the corresponding site in the UGT1A9 promoter prevented the binding of complex B and reduced promoter activity. Although Sp1 was able to bind to the Sp1/initiator-like site, its binding was dispensable for promoter activity. However, the binding of Sp1 to a second Sp1 site 30 bp 5' to the Sp1/initiator-like site greatly enhanced the activity of the UGT1A8 and 1A10 promoters. These results provide evidence that the UGT1A8, 1A9, and 1A10 genes are differentially regulated through an initiator element in their 5'-flanking regions.


Assuntos
Regulação da Expressão Gênica , Glucuronosiltransferase/genética , Regiões Promotoras Genéticas , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Clonagem Molecular , Primers do DNA/química , Sistema Digestório/metabolismo , Glucuronosiltransferase/química , Humanos , Rim/metabolismo , Fígado/metabolismo , Luciferases/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Transcrição Gênica , UDP-Glucuronosiltransferase 1A
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