RESUMO
The function of the peripheral cannabinoid receptor (CB2), which is mainly expressed on hematopoietic cells, remains an enigma. In an attempt to decipher its role, we used Affymetrix DNA chips to investigate the gene expression profile of the promyelocytic cells HL-60 transfected with the CB2 receptor and activated with the cannabinoid agonist CP 55,940. Agonist exposure of these cells led to an activation of a mitogen-activated protein kinase cascade and a receptor desensitization, indicating a functional coupling of the transfected receptors. At the genomic level, activation of the CB2 receptors induced an up-regulation of nine genes involved in cytokine synthesis, regulation of transcription, and cell differentiation. A majority of them are under the control of the transcription factor NF-kappaB, whose nuclear translocation was demonstrated. Many features of the transcriptional events, reported here for the first time, appeared to be related to an activation of a cell differentiation program, suggesting that CB2 receptors could play a role in the initialization of cell maturation. Moreover, we showed that CB2-activated wild-type HL-60 cells developed properties usually found in host defense effector cells such as an enhanced release of chemotactic cytokines and an increased motility, characteristic of more mature cells of the granulocytic-monocytic lineage.
Assuntos
Receptores de Droga/metabolismo , Canfanos/farmacologia , Canabinoides/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Movimento Celular/efeitos dos fármacos , Cicloexanóis/farmacologia , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação/efeitos dos fármacos , Pirazóis/farmacologia , RNA Mensageiro/metabolismo , Receptores de Canabinoides , Receptores de Droga/agonistas , TransfecçãoRESUMO
Listeria monocytogenes is a pathogenic intracellular microorganism whose infection induces pleiotropic biological changes associated with host cell gene expression regulation. Here we define the gene expression profiles of the human promyelocytic THP1 cell line before and after L. monocytogenes infection. Gene expression was measured on a large scale via oligonucleotide microarrays with probe sets corresponding to 6,800 human genes. We assessed and discussed the reproducibility of the hybridization signatures. In addition to oligonucleotide arrays, we also performed the large scale gene expression measurement with two high-density membranes, assaying for 588 and 18,376 human genes, respectively. This work allowed the reproducible identification of 74 up-regulated RNAs and 23 down-regulated RNAs as a consequence of L. monocytogenes infection of THP1. The reliability of these data was reinforced by performing independent infections. Some of these detected RNAs were consistent with previous results, while some newly identified RNAs encode gene products that may play key roles in L. monocytogenes infection. These findings will undoubtedly enhance the understanding of L. monocytogenes molecular physiology and may help identify new therapeutic targets.
Assuntos
Regulação da Expressão Gênica , Listeria monocytogenes/fisiologia , Northern Blotting , Linhagem Celular , Humanos , Sondas de Oligonucleotídeos , Reprodutibilidade dos TestesRESUMO
Using the recently developed methodology of nucleic acid microarrays spotted with specific cDNAs probes belonging to different gene families, we showed for the first time that nanomolar concentrations of the cannabinoid ligand CP-55940 upregulated the expression of two different members of the chemokine gene family: the alpha-chemokine interleukin-8 (IL-8) and the beta-chemokine monocyte chemotactic protein-1 (MCP-1), in the promyelocytic cell line HL60 transfected with peripheral cannabinoid receptors (CB2). These genomic modulations observed on large-scale cDNA arrays were first confirmed by Northern blot studies. Furthermore, ELISA evaluations in culture supernatants indicated that the cannabinoid-induced activation of these two chemokine genes was followed by enhanced expression and secretion of the corresponding proteins. These upregulations initially observed in transfected HL60 cells overexpressing CB2 receptors, also occurred in normal non-transfected HL60 cells. The enhancement of IL-8 and MCP-1 gene transcription and protein production was shown to be pertussis toxin sensitive attesting that this phenomenon was a Gi protein-coupled receptor-mediated process as expected for cannabinoid receptors. More specifically, the abolition of the cannabinoid-induced effect by the specific CB2 antagonist SR 144528 indicated a strict peripheral cannabinoid-mediated process. Altogether, our data highlight a possible new function of peripheral cannabinoid receptors in the modulation of immune and inflammatory responses.
Assuntos
Quimiocina CCL2/genética , Quimiocinas CXC , Peptídeos e Proteínas de Sinalização Intercelular , Interleucina-8/genética , Receptores de Droga/metabolismo , Analgésicos/farmacologia , Northern Blotting , Canfanos/farmacologia , Quimiocina CCL4 , Quimiocina CXCL1 , Fatores Quimiotáticos/metabolismo , Cicloexanóis/farmacologia , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Substâncias de Crescimento/metabolismo , Células HL-60 , Humanos , Proteínas Inflamatórias de Macrófagos/metabolismo , Pirazóis/farmacologia , Receptores de Canabinoides , Receptores de Droga/antagonistas & inibidores , Fatores de Tempo , TransfecçãoRESUMO
Using a cytoplasmic domain of the peripheral benzodiazepine receptor (PBR) as a bait in the yeast two-hybrid system, we have isolated a cDNA encoding a new protein that specifically interacts with PBR. We named it PRAX-1, for peripheral benzodiazepine receptor-associated protein 1. PRAX-1 is a 1857-amino acid protein, the sequence of which was structurally unrelated to any known proteins. The gene encoding PRAX-1 is located in the q22-q23 region of the long arm of the human chromosome 17. The PRAX-1 mRNA is 7.5 kilobase pairs, predominantly expressed in the central nervous system, pituitary gland, and thymus. At the protein level, we found the PRAX-1 as a single 220-250-kDa protein in the brain and in many different human cell lines tested using specific antibody raised against PRAX-1. Parallel analysis of the PRAX-1 mRNA and protein expression performed in mouse and rat gave similar results. Immunocytochemistry analysis carried out to define the distribution of the PRAX-1 protein in the rat brain showed that PRAX-1 was prevalent in the mesolimbic system, specially abundant in the CA1 subfield of the hippocampus. Exhibiting several domains involved in protein-protein interaction (three proline-rich domains, three leucine-zipper motifs, and an Src homology region 3-like domain), the PRAX-1 may be looked upon as a new adaptator protein. We show that both the Src homology region 3-like domain and a proline-rich domain in PRAX-1 are required for the interaction with PBR. PRAX-1 is a cytoplasmic protein that also partially colocalizes with PBR in the mitochondria, as determined by confocal microscopy and Western blotting. Altogether our observations support a model of interaction implicating PBR and this newly described protein, PRAX-1. As being the first cytoplasmic protein associated with PBR, PRAX-1 is a new tool that opens new fields for exploring PBR biological roles.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cromossomos Humanos Par 17 , Receptores de GABA-A/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Encéfalo/metabolismo , Galinhas , Bandeamento Cromossômico , Clonagem Molecular , Sequência Consenso , Humanos , Hibridização in Situ Fluorescente , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , RatosRESUMO
Two subtypes of G-protein-coupled cannabinoid receptors have been identified to date: the CB1 central receptor subtype, which is mainly expressed in the brain, and the CB2 peripheral receptor subtype, which appears particularly abundant in the immune system. We investigated the expression of CB2 receptors in leukocytes using anti-CB2 receptor immunopurified polyclonal antibodies. We showed that peripheral blood and tonsillar B cells were the leukocyte subsets expressing the highest amount of CB2 receptor proteins. Dual-color confocal microscopy performed on tonsillar tissues showed a marked expression of CB2 receptors in mantle zones of secondary follicles, whereas germinal centers (GC) were weakly stained, suggesting a modulation of this receptor during the differentiation stages from virgin B lymphocytes to memory B cells. Indeed, we showed a clear downregulation of CB2 receptor expression during B-cell differentiation both at transcript and protein levels. The lowest expression was observed in GC proliferating centroblasts. Furthermore, we investigated the effect of the cannabinoid agonist CP55,940 on the CD40-mediated proliferation of both virgin and GC B-cell subsets. We found that CP55,940 enhanced the proliferation of both subsets and that this enhancement was blocked by the CB2 receptor antagonist SR 144528 but not by the CB1 receptor antagonist SR 141716. Finally, we observed that CB2 receptors were dramatically upregulated in both B-cell subsets during the first 24 hours of CD40-mediated activation. These data strongly support an involvement of CB2 receptors during B-cell differentiation.
Assuntos
Subpopulações de Linfócitos B/citologia , Receptores de Droga/fisiologia , Animais , Subpopulações de Linfócitos B/metabolismo , Antígenos CD40/fisiologia , Células CHO , Canfanos/farmacologia , Diferenciação Celular , Cricetinae , Cricetulus , Cicloexanóis/farmacologia , Centro Germinativo/citologia , Humanos , Microscopia Confocal , Tonsila Palatina/citologia , Fragmentos de Peptídeos/imunologia , Piperidinas/farmacologia , Pirazóis/farmacologia , Coelhos , Receptores de Canabinoides , Receptores de Droga/efeitos dos fármacos , Receptores de Droga/genética , Receptores de Droga/imunologia , Proteínas Recombinantes de Fusão/fisiologia , Rimonabanto , TransfecçãoRESUMO
SR 31747A, defined as a sigma ligand, is a novel immunosuppressive agent that blocks proliferation of human and mouse lymphocytes. Using a radiolabeled chemical probe, we here purified a target of SR 31747A and called it SR 31747A-binding protein (SR-BP). Purified SR-BP retained its binding properties and migrated on SDS-polyacrylamide gel as a Mr 28,000 protein. Cloning of the cDNA encoding human SR-BP shows an open reading frame for a 223-amino acid protein, which is homologous to the recently cloned sigma 1 receptor. Interestingly, the deduced amino acid sequence was found to be related to fungal C8-C7 sterol isomerase, encoded by the ERG2 gene. The ERG2 gene product has been identified recently as the molecular target of SR 31747A that mediates antiproliferative effects of the drug in yeast. Northern blot analysis of SR-BP gene expression revealed a single transcript of 2 kilobases which was widely expressed among organs, with the highest abundance in liver and the lowest abundance in brain. Subcellular localization analysis in various cells, using a specific monoclonal antibody raised against SR-BP, demonstrated that this protein was associated with the nuclear envelope. When studying the binding of SR 31747A on membranes from yeast expressing SR-BP, we found a pharmacological profile of sigma 1 receptors; binding was displaced by (+)-pentazocine, haloperidol, and (+)-SKF 10,047, with (+)-SKF 10, 047 being a more potent competitor than (-)-SKF 10,047. Scatchard plot analysis revealed Kd values of 7.1 nM and 0.15 nM for (+)-pentazocine and SR 31747A, respectively, indicating an affinity of SR-BP 50-fold higher for SR 31747A than for pentazocine. Additionally, we showed that pentazocine, a competitive inhibitor of SR 31747A binding, also prevents the immunosuppressive effect of SR 31747A. Taken together, these findings strongly suggest that SR-BP represents the molecular target for SR 31747A in mammalian tissues, which could be critical for T cell proliferation.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Cicloexanos/metabolismo , Proteínas de Ligação a DNA/química , Receptores Opioides , Saccharomyces cerevisiae/metabolismo , Esteroide Isomerases/química , Transativadores/química , Sequência de Aminoácidos , Animais , Anti-Inflamatórios não Esteroides/metabolismo , Sequência de Bases , Ligação Competitiva , Proteínas de Transporte/isolamento & purificação , Membrana Celular/metabolismo , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia por Troca Iônica , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go , Humanos , Cinética , Camundongos , Dados de Sequência Molecular , Receptores sigma/química , Receptores sigma/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Esteroide Isomerases/metabolismo , Linfócitos T , Transativadores/metabolismo , Regulador Transcricional ERG , Receptor Sigma-1RESUMO
Cholinesterase inhibitors occur naturally in the calabar bean (eserine), green potatoes (solanine), insect-resistant crab apples, the coca plant (cocaine) and snake venom (fasciculin). There are also synthetic cholinesterase inhibitors, for example man-made insecticides. These inhibitors inactivate acetylcholinesterase and butyrylcholinesterase as well as other targets. From a study of the tissue distribution of acetylcholinesterase and butyrylcholinesterase mRNA by Northern blot analysis, we have found the highest levels of butyrylcholinesterase mRNA in the liver and lungs, tissues known as the principal detoxication sites of the human body. These results indicate that butyrylcholinesterase may be a first line of defense against poisons that are eaten or inhaled.
Assuntos
Acetilcolinesterase/genética , Butirilcolinesterase/genética , RNA Mensageiro/metabolismo , Acetilcolinesterase/sangue , Acetilcolinesterase/metabolismo , Animais , Northern Blotting , Encéfalo/metabolismo , Butirilcolinesterase/sangue , Butirilcolinesterase/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Rim/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Camundongos , Peso Molecular , Músculo Esquelético/metabolismo , Músculos/metabolismo , Miocárdio/metabolismo , Pâncreas/metabolismo , Placenta/metabolismo , RNA Mensageiro/genética , Coelhos , Ratos , Baço/metabolismo , Distribuição TecidualRESUMO
Comparison of proteins expressed by SV40 transformed cell lines and untransformed cell lines is of interest because SV40 transformed cells are immortal, whereas untransformed cells senesce after about 50 doublings. In MRC-5 SV40 cells, only seven proteins have previously been reported to shift from undetectable to detectable after transformation by SV40 virus. We report that butyrylcholinesterase is an 8th protein in this category. Butyrylcholinesterase activity in transformed MRC-5 SV40 cells increased at least 150-fold over its undetectable level in MRC-5 parental cells. Other SV40 transformed cell lines, including COS-1, COS-7, and WI-38 VA13, also expressed endogenous butyrylcholinesterase, whereas the parental, untransformed cell lines, CV-1 and WI-38, had no detectable butyrylcholinesterase activity or mRNA. Infection of CV-1 cells by SV40 virus did not result in expression of butyrylcholinesterase, showing that the butyrylcholinesterase promoter was not activated by the large T antigen of SV40. We conclude that butyrylcholinesterase expression resulted from events related to cell immortalization and did not result from activation by the large T antigen.
Assuntos
Butirilcolinesterase/metabolismo , Transformação Celular Viral , Vírus 40 dos Símios , Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Animais , Northern Blotting , Butirilcolinesterase/genética , Carcinoma Hepatocelular/enzimologia , Linhagem Celular Transformada , Chlorocebus aethiops , Embrião de Mamíferos , Humanos , Técnicas de Imunoadsorção , Rim , Neoplasias Hepáticas/enzimologia , Pulmão , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
A large cDNA fragment covering the complete sequence of the mature catalytic subunit of rabbit acetylcholinesterase (AChE) has been cloned and sequenced. This sequence was compared to that of rabbit butyrylcholinesterase [BChE; Jbilo, O. & Chatonnet, A. (1990) Nucleic Acids Res. 18, 3990]. Amino acid sequences of AChE and BChE have 51% identity. They both possessed a choline-binding site W84, a catalytic triad S200-H440-E327 and six cysteine residues (positions 67-94, 254-265, 402-521) in conserved sequence positions to those that form three intrachain disulfide bonds in all cholinesterases (by convention, numbering of amino acids is that used for Torpedo AChE). Rabbit AChE had a larger number of aromatic residues lining the active-site gorge than rabbit BChE (14 compared to 8, respectively) and a smaller number of potential N-glycosylation sites (3 compared to 8, respectively). Both catalytic subunits have a hydrophilic C-terminus (catalytic subunits of type T). Expression of acetylcholinesterase and butyrylcholinesterase genes (ACHE and BCHE) was studied in rabbit tissues and during development by a correlation of Northern-blot analysis and enzymic activities. This correlation was rendered difficult by the presence of an eserine-resistant esterase active on butyrylthiocholine in serum, liver and lung. When the contribution of this carboxylesterase was taken into account, brain was found as the richest source of BChE followed by lung and heart. Rabbit liver had a very low content of BChE that correlated with the low BChE activity in plasma. During development, BCHE transcripts were detected as early as day 10 post coitum, whereas ACHE transcripts appeared only on day 12.
Assuntos
Acetilcolinesterase/biossíntese , Envelhecimento/metabolismo , Butirilcolinesterase/biossíntese , Regulação Enzimológica da Expressão Gênica , Acetilcolinesterase/sangue , Acetilcolinesterase/isolamento & purificação , Sequência de Aminoácidos , Animais , Northern Blotting , Encéfalo/enzimologia , Encéfalo/crescimento & desenvolvimento , Butirilcolinesterase/sangue , Butirilcolinesterase/isolamento & purificação , Clonagem Molecular , DNA Complementar , Desenvolvimento Embrionário e Fetal , Biblioteca Gênica , Coração/crescimento & desenvolvimento , Humanos , Isoenzimas/biossíntese , Isoenzimas/isolamento & purificação , Pulmão/enzimologia , Pulmão/crescimento & desenvolvimento , Dados de Sequência Molecular , Miocárdio/enzimologia , Especificidade de Órgãos , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Coelhos , Homologia de Sequência de AminoácidosRESUMO
Two kilobase segments of the 5'-untranslated regions of the human and rabbit butyrylcholinesterase (BCHE) genes were characterized. The sequences shared extensive identity except for a 333-base pair (bp) Alu repeat present only in human BCHE. One single transcription start site was found in both genes with the techniques of primer extension, amplification of the 5'-end of mRNA, and RNase protection. Cap sites in human and rabbit BCHE genes were found in strictly homologous positions. In human BCHE, the transcription start site was found 157 bp upstream of Met-28, the translation start site. Potential regulatory elements in both promoters included one AP1 site and multiple sites for topoisomerase, Oct-1 and PEA-3. Transient expression of BCHE-reporter gene constructs showed that a 194-bp fragment of the 5'-flanking region of human BCHE and a 570-bp fragment of rabbit BCHE were sufficient for promoting chloramphenicol acetyltransferase activity in HeLa cells. No consensus TATA and CAAT boxes were found. However, the sequence around the transcription start site exhibited homology with initiator elements found in other TATA-less promoters in developmentally regulated genes.
Assuntos
Butirilcolinesterase/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Cloranfenicol O-Acetiltransferase/genética , DNA/química , DNA/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/metabolismo , Coelhos , Alinhamento de SequênciaRESUMO
Histoenzymatic methods and in situ hybridization were used to follow AChE expression in rabbit embryos from 10 to 15 days. Transcripts of AChE are detected at the same developmental stages in all structures where enzymatic activity is found, except in neuronal extension and the ventral part of mesonephros. AChE and BChE expression were compared. BChE transcripts are detected before BChE activity can be revealed in blood cells and mesonephros.
Assuntos
Acetilcolinesterase/metabolismo , Embrião de Mamíferos/enzimologia , RNA Mensageiro/metabolismo , Acetilcolinesterase/análise , Acetilcolinesterase/biossíntese , Animais , Butirilcolinesterase/análise , Butirilcolinesterase/biossíntese , Butirilcolinesterase/metabolismo , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário e Fetal , Idade Gestacional , Hibridização In Situ , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , CoelhosRESUMO
We investigated the myogenic properties and the expression of acetylcholinesterase (AChE) in culture of satellite cells (SCs) isolated from slow and fast rabbit muscles. Slow SCs form myotubes more rapidly (day 9 vs day 11) than fast SCs, and differentiate further into striated and contractile fibers. AChE activity and mRNA expression are higher in SCs cultured from slow than from fast muscles, as also observed in the muscles themselves. However, the two types of SC cultures do not show obvious difference in their patterns of AChE molecular forms. Taken together, these preliminary data support the view that there might be more than one SC population in skeletal muscles.
Assuntos
Acetilcolinesterase/biossíntese , Regulação Enzimológica da Expressão Gênica , Isoenzimas/biossíntese , Músculos/enzimologia , Acetilcolinesterase/genética , Acetilcolinesterase/isolamento & purificação , Animais , Northern Blotting , Diferenciação Celular , Células Cultivadas , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Músculos/citologia , Polimorfismo Genético , RNA Mensageiro/biossíntese , RNA Mensageiro/isolamento & purificação , CoelhosRESUMO
1. Three clones were isolated from a rabbit genomic library. They covered the entire coding sequence of the rabbit BChE gene. The positions of splice sites between exons 2, 3, and 4 are identical to those found in the human gene (Arpagaus et al., 1990). Exon 2 covers 83% of the coding sequence. This contrasts with the small size of exon 3 (167 bp) and large size of introns 2 and 3 (greater than 20 kb each). The active-site serine at position 198 is found in a highly conserved region. Aspartic acids in positions 91 and 170 are conserved in human and rabbit, and one of them could be involved in the calytic triad. Aspartic acid 70, present in the anionic site of human BChE, is also conserved in rabbit BChE. The coding sequences of human and rabbit BChE are 89% identical over 744 bp around the active-site serine. 2. In addition to the genomic clones, one cDNA clone (BNY1) was isolated. This cDNA was unusual in that it contained intronic sequences. The insert of 1 kb contained 167 coding bases homologous to the nucleotide sequence 1434 to 1600 of human cDNA and corresponded to exon 3 of the BChE gene. On each side of this coding region, consensus sequences of intron-exon boundaries were found. 3. The presence of large-size transcripts in Northern blots and the existence of a cDNA copy of unprocessed mRNA found in the BNY1 clone suggest a slow processing of transcripts. A genomic sequence unspliced in a cDNA of Torpedo AChE could give a transmembrane domain (Sikorav et al., 1988); the corresponding sequence in rabbit BChE gene, also found in a cDNA, had no homology with Torpedo AChE but could be translated in a hydrophobic C-terminal domain if maintained in mature mRNA.