RESUMO
Prostate cancer (PCa) is one of the major public health problems in Western countries. Recently, the TMPRSS2:ERG gene fusion, which results in the aberrant expression of the transcription factor ERG, has been shown to be the most common gene rearrangement in PCa. Previous studies have determined the contributions of this fusion in PCa disease initiation and/or progression in vitro and in vivo. In this study on TMPRSS2:ERG regulation in PCa, we used an androgen receptor and TMPRSS2:ERG fusion double-negative PCa cell model: PC3c. In three cell clones with different TMPRSS2:ERG expression levels, ectopic expression of the fusion resulted in significant induction of cell migration and invasion in a dose-dependent manner. In agreement with this phenotype, high-throughput microarray analysis revealed that a set of genes, functionally associated with cell motility and invasiveness, were deregulated in a dose-dependent manner in TMPRSS2:ERG-expressing cells. Importantly, we identified increased MMP9 (Metalloproteinase 9) and PLXNA2 (Plexin A2) expression in TMPRSS2:ERG-positive PCa samples, and their expression levels were significantly correlated with ERG expression in a PCa cohort. In line with these findings, there was evidence that TMPRSS2:ERG directly and positively regulates MMP9 and PLXNA2 expression in PC3c cells. Moreover, PLXNA2 upregulation contributed to TMPRSS2:ERG-mediated enhancements of PC3c cell migration and invasion. Furthermore, and importantly, PLXNA2 expression was upregulated in metastatic PCa tumors compared with localized primary PCa tumors. This study provides novel insights into the role of the TMPRSS2:ERG fusion in PCa metastasis.
Assuntos
Metaloproteinase 9 da Matriz/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Neoplasias da Próstata/metabolismo , Receptores de Superfície Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Metástase Linfática , Masculino , Proteínas de Fusão Oncogênica/genética , Fenótipo , Neoplasias da Próstata/patologia , TranscriptomaRESUMO
BACKGROUND: Inevitable hepatitis C virus recurrence after liver transplantation, a major barrier to survival of the transplanted liver may be promoted by immunosuppression and by CD4(+)CD25(+) regulatory T cells (Treg). Treg cells are essential for the induction and maintenance of immunologic self-tolerance as well as transplant tolerance. Moreover, we have previously described low doses of cyclosporine (CsA) to inhibit Treg activity by inducing interleukin-2 and interfron-γ. We investigated here in, the effect of mycophenolate mofetil (MMF) and corticosteroids, usually used in combination with a calcineurin inhibitor on human CD4(+)CD25(+) Treg cells. METHODS: Human CD4(+)CD25(+) cells isolated from healthy donors were cultured in the presence of CsA +/- corticoids or MMF. Suppressive activity of regulatory T cells was assessed in mixed leukocyte reactions including CD25(+) solvents with autologous activated peripheral blood mononuclear cells (PBMC). RESULTS: MMF and dexamethasone inhibited PBMC and Treg proliferation in dose-dependent fashing, maintaining the suppressive activity of Treg cells. However, the association of corticoids with CsA could not reverse the inhibitory effects of CsA on Treg activity, unlike the MMF and CsA combination. CONCLUSION: We have previously shown CsA to significantly impair the function of CD4(+)CD25(+) Treg cells. Herein we reports that corticoids were not able to reverse this effect, whereas MMF couterbalanced it, suggesting that the combination of MMF with CsA maintains regulatory T cells activity promoting tolerance.
Assuntos
Corticosteroides/farmacologia , Ciclosporina/farmacologia , Dexametasona/farmacologia , Imunossupressores/farmacologia , Ácido Micofenólico/análogos & derivados , Linfócitos T Reguladores/efeitos dos fármacos , Biomarcadores/metabolismo , Calcineurina/metabolismo , Inibidores de Calcineurina , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Humanos , Tolerância Imunológica/efeitos dos fármacos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Teste de Cultura Mista de Linfócitos , Ácido Micofenólico/farmacologia , Linfócitos T Reguladores/imunologiaRESUMO
In this study, we report that the PEA3 group members interact with the mammalian really interesting new gene (RING) E3 ubiquitin ligase constitutive photomorphogenetic 1 (COP1), which mediates ubiquitylation and subsequent proteasome degradation of the p53 and c-Jun transcription factors. This interaction is mediated by the central region of COP1 including the coiled-coil domain and two COP1-interacting consensus motifs localized in the well-conserved N-terminal transactivation domain of the PEA3 group members. At the transcriptional level, COP1 reduces the transcriptional activity of ERM and the two other PEA3 group proteins on Ets-responsive reporter genes; this effect being dependent on the RING domain of COP1 and the two COP1-interacting motifs of ERM. Reduced transcriptional activity was, however, not related to COP1-induced changes in ERM stability. In fact, increased ubiquitylation and subsequent proteasome-mediated degradation of ERM is achieved only when COP1 is expressed with DET1, a key COP1 partner within the ubiquitylation complex. Conversely, we show that the depletion of COP1 or DET1 by small interference RNA (siRNA) in U2OS cells stabilizes endogenous ERM whereas only COP1 knockdown enhances expression of ICAM-1, a gene regulated by this transcription factor. These results indicate that COP1 is a complex regulator of ERM and the two other PEA3 group members.
Assuntos
Neoplasias/genética , Transativadores/metabolismo , Transcrição Gênica , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Sequência Conservada , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Variação Genética , Homeostase , Humanos , Dados de Sequência Molecular , Interferência de RNA , RNA Interferente Pequeno/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genéticaRESUMO
From the conditioned medium of the human colon carcinoma cells, HT-29 5M21 (CM-5M21), expressing a spontaneous invasive phenotype, tumor-associated trypsin inhibitor (TATI) was identified and characterized by proteomics, cDNA microarray approaches and functional analyses. Both CM-5M21 and recombinant TATI, but not the K18Y-TATI mutant at the protease inhibitor site, trigger collagen type I invasion by several human adenoma and carcinoma cells of the colon and breast, through phosphoinositide-3-kinase, protein kinase C and Rho-GTPases/Rho kinase-dependent pathways. Conversely, the proinvasive action of TATI in parental HT29 cells was alleviated by the TATI antibody PSKAN2 and the K18Y-TATI mutant. Stable expression of K18Y-TATI in HT-29 5M21 cells downregulated tumor growth, angiogenesis and the expression of several metastasis-related genes, including CSPG4 (13.8-fold), BMP-7 (9.7-fold), the BMP antagonist CHORDIN (5.2-fold), IGFBP-2 and IGF2 (9.6- and 4.6-fold). Accordingly, ectopic expression of KY-TATI inhibited the development of lung metastases from HT-29 5M21 tumor xenografts in immunodeficient mice. These findings identify TATI as an autocrine transforming factor potentially involved in early and late events of colon cancer progression, including local invasion of the primary tumor and its metastatic spread. Targeting TATI, its molecular partners and effectors may bring novel therapeutic applications for high-grade human solid tumors in the digestive and urogenital systems.
Assuntos
Comunicação Autócrina , Neoplasias do Colo/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Inibidor da Tripsina Pancreática de Kazal/metabolismo , Adenoma/genética , Adenoma/metabolismo , Adenoma/patologia , Animais , Anticorpos Antineoplásicos/farmacologia , Comunicação Autócrina/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Camundongos , Camundongos SCID , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/genética , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Inibidor da Tripsina Pancreática de Kazal/genética , Inibidor da Tripsina Pancreática de Kazal/farmacologiaRESUMO
To regulate the spatiotemporal expression of their target genes, the transcription factors undergo post-translational modifications of which the most studied is phosphorylation. Acetylation and ubiquitinylation on lysine residues also exert a role in the transcription, as it is the case for the regulation of the activity of the huge family of Ets transcription factors. Recently, sumoylation, a post-translational modification similar to ubiquitinylation, was described as playing a crucial role in the inhibition of the activity of these factors.
Assuntos
Regulação da Expressão Gênica , Processamento de Proteína Pós-Traducional , Proteína Proto-Oncogênica c-ets-1/metabolismo , Proteína SUMO-1/metabolismo , Ubiquitina/metabolismo , Acetilação , Animais , Modelos Animais de Doenças , Humanos , Leucemia/genética , Leucemia/metabolismo , Lisina/metabolismo , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Fosforilação , Proteína Proto-Oncogênica c-ets-1/genéticaRESUMO
ERM is a member of the ETS transcription factor family. High levels of the corresponding mRNA are detected in a variety of human breast cancer cell lines, as well as in aggressive human breast tumors. As ERM protein is almost undetectable in these cells, high degradation of this transcription factor has been postulated. Here we have investigated whether ERM degradation might depend on the proteasome pathway. We show that endogenous and ectopically expressed ERM protein is short-lived protein and undergoes proteasome-dependent degradation. Deletion mutagenesis studies indicate that the 61 C-terminal amino acids of ERM are critical for its proteolysis and serve as a degradation signal. Although ERM conjugates with ubiquitin, this post-translational modification does not depend on the C-terminal domain. We have used an Ets-responsive ICAM-1 reporter plasmid to show that the ubiquitin-proteasome pathway can affect transcriptional function of ERM. Thus, ERM is subject to degradation via the 26S proteasome pathway, and this pathway probably plays an important role in regulating ERM transcriptional activity.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Western Blotting , Neoplasias da Mama/metabolismo , Células COS , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Humanos , Imunoprecipitação , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Rim/metabolismo , Mutagênese Sítio-Dirigida , Plasmídeos , Processamento de Proteína Pós-Traducional , Coelhos , Fatores de Transcrição/genética , Ativação Transcricional , Células Tumorais Cultivadas , Ubiquitina/metabolismoRESUMO
Small DNA tumour viruses have evolved a number of mechanisms to drive nondividing cells into S phase. Virally encoded oncoproteins such as adenovirus E1A and human papillomavirus (HPV) E7 can bind an array of cellular proteins to override proliferation arrest. The DNA methyltransferase Dnmt1 is the major mammalian enzyme responsible for maintaining CpG methylation patterns in the cell following replication. One of the hallmarks of tumour cells is disrupted DNA methylation patterns, highlighting the importance of the proper regulation of DNA methyltransferases in normal cell proliferation. Here, we show that adenovirus 5 E1A and HPV-16 E7 associate in vitro and in vivo with the DNA methyltransferase Dnmt1. Consistent with this interaction, we find that E1A and E7 can purify DNA methyltransferase activity from nuclear extracts. These associations are direct and mediated by the extreme N-terminus of E1A and the CR3 zinc-finger domain of E7. Furthermore, we find that a point mutant at leucine 20 of E1A, a residue known to be critical for its transformation functions, is unable to bind Dnmt1 and DNA methyltransferase activity. Finally, both E1A and E7 can stimulate the methyltransferase activity of Dnmt1 in vitro. Our results provide the first indication that viral oncoproteins bind and regulate Dnmt1 enzymatic activity. These observations open up the possibility that this association may be used to control cellular proliferation pathways and suggest a new mechanism by which small DNA tumour viruses can steer cells through the cell cycle.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Linhagem Celular , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA , Papillomavirus Humano 16/metabolismo , HumanosRESUMO
The incidence of prostate cancer is increasing in western countries because of population aging. Prostate cancer begins as an androgen-dependent disease, but it can become androgen independent at a later stage or in tumors recurring after an antihormonal treatment. Although many genetic events have been described to be involved in androgen-dependent and/or -independent prostate cancer growth, little is known about the contribution of epigenetic events. Here we have examined the possibility that the methyl-CpG-binding protein MECP2 might play a role in controlling the growth of prostate cancer cells. Inhibition of MECP2 expression by stable short hairpin RNA stopped the growth of both normal and cancer human prostate cells. In addition, ectopic expression of the MECP2 conferred a growth advantage to human prostate cancer cells. More importantly, this expression allowed androgen-dependent cells to grow independently of androgen stimulation and to retain tumorigenic properties in androgen-depleted conditions. Analysis of signaling pathways showed that this effect is independent of androgen receptor signaling. Instead, MECP2 appears to act by maintaining a constant c-myc level during antihormonal treatment. We further show that MECP2-expressing cells possess a functional p53 pathway and are still responsive to chemotherapeutic drugs.
Assuntos
Proliferação de Células , Proteína 2 de Ligação a Metil-CpG/fisiologia , Neoplasias da Próstata/patologia , Androgênios/fisiologia , Antineoplásicos Hormonais/farmacologia , Sobrevivência Celular , Humanos , Masculino , Próstata/citologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores Androgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Células Tumorais CultivadasRESUMO
B19 may cause mild to severe clinical manifestations. Owing to the remarkable tropism of B19 for red blood cell progenitors, there is a lack of satisfactory cell lines fully permissive for B19. Because the local oxygen pressure may influence viral replication, we used hypoxia to improve the sensitivity of our infectivity assay in order to link B19 DNA detected by PCR to the presence of infectious B19 particles in plasma. Plasma samples and the WHO International Standard for B19 DNA detection by PCR were used to infect the pluripotent human erythroid cell line KU812F under different oxygen pressures. Specific human anti-B19 IgG was found to reduce infectivity. Low oxygen pressure led to higher yields of infectious B19 progeny and to a higher level of viral transcription than observed under normoxia. This sensitive infectivity assay is a promising model for studying B19 biology, identifying neutralising antibodies, and evaluating new virus inactivation methods.
Assuntos
Hipóxia Celular , Parvovirus B19 Humano/crescimento & desenvolvimento , Proteínas do Capsídeo/antagonistas & inibidores , Linhagem Celular , Células Precursoras Eritroides , Eritropoetina , Humanos , Imunoglobulina G/farmacologia , Parvovirus B19 Humano/patogenicidade , Fatores de Tempo , Cultura de Vírus/métodosRESUMO
erm, er81 and pea3 are three related genes that define a novel Ets-related subfamily of transcription factors. The expression patterns of these genes has been previously characterized in the mouse from embryonic day (E) 9.5 to birth (Oncogene 15 (1997) 937). In this study, we report differential expression patterns of the PEA3 group genes during early mouse post-implantation development. erm and pea3 expression patterns were partly overlapping. erm was activated prior to pea3 in the distal tip of the embryonic epiblast but, at primitive streak-stages, both genes were coexpressed in the posterior region of the embryo in epiblast, primitive streak and adjacent mesoderm. Similar erm and pea3 expression patterns were seen later in posterior neural plate, presomitic and lateral mesoderm, mesonephros, and tail bud. Only erm, however, was expressed in specific brain regions corresponding to prospective midbrain and ventral forebrain. erm was also strongly expressed as early as E8 in the developing branchial region, especially at the level of branchial pouches, whereas pea3 transcripts appeared later in frontonasal and first arch mesenchyme. er81 transcripts were not detected prior to E9.0-9.5, suggesting that this gene may not be involved in early developmental events.
Assuntos
Desenvolvimento Embrionário e Fetal/genética , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genética , Animais , Encéfalo/embriologia , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Camundongos , Proteínas Proto-Oncogênicas c-etsRESUMO
The ets genes encode eukaryotic transcription factors that are involved in tumorigenesis and developmental processes. The signature of the Ets family is the ETS-domain, which binds to sites containing a central 5'-GGAA/T-3' motif. They can be sub-classified primarily because of the high amino acid conservation in their ETS-domains and, in addition, in the conservation of other domains generally characterized as transactivating. This is the case for the PEA3 group, which is currently made up of three members, PEA3/E1AF, ER81/ETV1 and ERM, which are more than 95% identical in the ETS-domain and more than 85% in the transactivation acidic domain. The members of the PEA3 group are activated through both the Ras-dependent and other kinase pathways, a function which emphasizes their involvement in several oncogenic mechanisms. The expression pattern of the three PEA3 group genes during mouse embryogenesis suggests that they are differentially regulated, probably to serve important functions such as tissue interaction. Although the target genes of these transcription factors are multiple, their most frequently studied role concerns their involvement in the metastatic process. In fact, PEA3 group members are over-expressed in metastatic human breast cancer cells and mouse mammary tumors, a feature which suggests a function of these transcription factors in mammary oncogenesis. Moreover, when they are ectopically over-expressed in non-metastatic breast cancer cells, these latter become metastatic with the activation of transcription of matrix metalloproteinases or adhesion molecules, such as ICAM-1.
Assuntos
Neoplasias da Mama/genética , Neoplasias Mamárias Animais/genética , Fatores de Transcrição/genética , Animais , Neoplasias da Mama/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Mamárias Animais/patologia , Camundongos , Invasividade Neoplásica/genéticaRESUMO
The Ets transcription factors of the PEA3 group--E1AF/PEA3, ETV1/ER81 and ERM--are almost identical in the ETS DNA-binding and the transcriptional acidic domains. To accelerate our understanding of the molecular basis of putative diseases linked to ETV1 such as Ewing's sarcoma we characterized the human ETV1 and the mouse ER81 genes. We showed that these genes are both encoded by 13 exons in more than 90 kbp genomic DNA, and that the classical acceptor and donor splicing sites are present in each junction except for the 5' donor site of intron 9 where GT is replaced by TT. The genomic organization of the ETS and acidic domains in the human ETV1 and mouse ER81 (localized to chromosome 12) genes is similar to that observed in human ERM and human E1AF/PEA3 genes. Moreover, as in human ERM and human E1AF/PEA3 genes, a first untranslated exon is upstream from the first methionine, and the mouse ER81 gene transcription is regulated by a 1.8 kbp of genomic DNA upstream from this exon. In human, the alternative splicing of the ETV1 gene leads to the presence (ETV1 alpha) or the absence (ETV1 beta) of exon 5 encoding the C-terminal part of the transcriptional acidic domain, but without affecting the alpha helix previously described as crucial for transactivation. We demonstrated here that the truncated isoform (human ETV1 beta) and the full-length isoform (human ETV1 alpha) bind similarly specific DNA Ets binding sites. Moreover, they both activate transcription similarly through the PKA-transduction pathway, so suggesting that this alternative splicing is not crucial for the function of this protein as a transcription factor. The comparison of human ETV1 alpha and human ETV1 beta expression in the same tissues, such as the adrenal gland or the bladder, showed no clear-cut differences. Altogether, these data open a new avenue of investigation leading to a better understanding of the functional role of this transcription factor.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Genes , Isoformas de Proteínas/genética , Splicing de RNA , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patologia , Mapeamento Cromossômico , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/química , Éxons/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Ligação Proteica , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/química , Estrutura Terciária de Proteína , Coelhos , Especificidade da Espécie , Fatores de Transcrição/biossíntese , Fatores de Transcrição/química , Transcrição Gênica , Células Tumorais CultivadasRESUMO
The E1AF protein belongs to the family of Ets transcription factors and is involved in the regulation of metastasis gene expression. It has recently been reported in an undifferentiated child sarcoma that part of this gene could be fused by translocation to the ews gene. We show here that the human e1af gene, which is located in the q21 region of chromosome 17, is organized in 13 exons distributed along 19kb of genomic DNA. Its two main functional domains, the acidic domain and the DNA-binding ETS domain, are each encoded by three different exons. The 3'-untranslated region of e1af is 0.7kb. The 5'-untranslated region is about 0.3kb and is composed of a first exon upstream from the exon containing the first methionine. These data could possibly accelerate an understanding of the molecular basis of putative inherited diseases linked to E1AF.
Assuntos
Proteínas E1A de Adenovirus/genética , Genes/genética , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Linhagem Celular , DNA/química , DNA/genética , Éxons , Regulação da Expressão Gênica , Células HeLa , Humanos , Íntrons , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-ets , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Células Tumorais CultivadasRESUMO
The HSD17B4 gene codes for a 80 kDa multifunctional enzyme containing three distinct functional domains and is localized in peroxisomes. The N-terminal part exhibits 3-hydroxyacyl-CoA dehydrogenase and 17beta-hydroxysteroid dehydrogenase activity whereas the central part shows enoyl-CoA hydratase activity. The carboxy-terminal part of the protein has sterol-carrier-protein activity. The protein is widely expressed, however in several tissues like brain, uterus and lung its expression is limited to specific cells like Purkinje cells or luminal epithelium. The HSD17B4 gene consist of 24 exons and 23 introns with classical intron-exon junctions spanning more than 100 kbp. The importance of the HSD17B4 protein is stressed by the identification of patients with severe clinical abnormalities due to mutations in the HSD17B4 gene. We have now checked the consequences of one frequent mutation, G16 S, which results in inactivation of the enzyme due to loss of interaction with NAD+.
Assuntos
17-Hidroxiesteroide Desidrogenases/genética , Enoil-CoA Hidratase , Complexos Multienzimáticos , 17-Hidroxiesteroide Desidrogenases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Éxons , Humanos , Hidroliases , Imuno-Histoquímica , Íntrons , Mutagênese , Proteína Multifuncional do Peroxissomo-2 , RNA Mensageiro/genética , SuínosRESUMO
Six types of human 17beta-hydroxysteroid dehydrogenases catalyzing the conversion of estrogens and androgens at position C17 have been identified so far. The peroxisomal 17beta-hydroxysteroid dehydrogenase type 4 (17beta-HSD 4, gene name HSD17B4) catalyzes the oxidation of estradiol with high preference over the reduction of estrone. The highest levels of 17beta-HSD 4 mRNA transcription and specific activity are found in liver and kidney followed by ovary and testes. A 3 kb mRNA codes for an 80 kDa (737 amino acids) protein featuring domains which are not present in the other 17beta-HSDs. The N-terminal domain of 17beta-HSD 4 reveals only 25% amino acid similarity with the other types of 17beta-HSDs. The 80 kDa protein is N-terminally cleaved to a 32 kDa enzymatically active fragment. Both the 80 kDa and the N-terminal 32 kDa (amino acids 1-323) protein are able to perform the dehydrogenase reaction not only with steroids at the C17 position but also with D-3-hydroxyacyl-coenzyme A (CoA). The enzyme is not active with L-stereoisomers. The central part of the 80 kDa protein (amino acids 324-596) catalyzes the 2-enoyl-acyl-CoA hydratase reaction with high efficiency. The C-terminal part of the 80 kDa protein (amino acids 597-737) facilitates the transfer of 7-dehydrocholesterol and phosphatidylcholine between membranes in vitro. The HSD17B4 gene is stimulated by progesterone, and ligands of PPARalpha (peroxisomal proliferator activated receptor alpha) such as clofibrate, and is down-regulated by phorbol esters. Mutations in the HSD17B4 lead to a fatal form of Zellweger syndrome.
Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Enoil-CoA Hidratase/metabolismo , Complexos Multienzimáticos , Síndrome de Zellweger/enzimologia , 17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/isolamento & purificação , Sequência de Aminoácidos , Animais , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Humanos , Hidroliases , Dados de Sequência Molecular , Mutação , Proteína Multifuncional do Peroxissomo-2 , Homologia de Sequência de Aminoácidos , Frações Subcelulares/enzimologia , SuínosRESUMO
Microsomal cytochrome P450c17 (17a-hydroxylase/17,20-Lyase) catalyzes two reactions in the delta5 and delta4 pathways leading to the production of C19 steroids. Transient expression of human, bovine, porcine, rat, and mouse P450c17 cDNAs showed that the protein has 17alpha-hydroxylase and 17,20-Lyase activities, converting pregnenolone and progesterone into delta5- and delta4-Cl9 steroids, respectively, although the rat and mouse proteins have a preferential pathway toward the delta4 steroids. The guinea pig (gp) P450c17 shares 46% to 70% amino acid identity with the corresponding proteins of other species, and further characterization indicated that the guinea pig enzyme only converts progesterone to androstenedione. In this study, we have tried to identify amino acid(s) in the gpP450c17 that governs such a steroid specificity. Among the various mutants that we have created, change of the arginine (R) residue at position 200 to an asparagine (N) (R200N) in the gpP450c17 protein increased reactivity toward pregnenolone compared with the wild-type enzyme. Pregnenolone was converted into 17alpha-hydroxypregnenolone and dehydroepiandrosterone. However, this gain occurred at the expense of the 17,20-lyase activity toward 17alpha-hydroxyprogesterone. The R200N mutation in the gpP450c17 protein introduced a potential N-linked glycosylation site (200Asn-X-Thr202); however, substitution of the Thr202 residue by an asparagine (R200N/T202N), which abolishes the site, did not change the preference of the gpP450c17 mutant for pregnenolone. Furthermore, introduction of a putative glycosylation site at amino acid 185 in the gpP450c17 enzyme did not alter substrate specificity. The properties of the amino acid were also investigated, and neither the charge nor the size of the sidechain elicited change in the substrate specificity of gpP450c17. Thus, our results demonstrate that the mutation of arginine to asparagine at position 200 changes the substrate specificity of the gpP450c17 enzyme.
Assuntos
Complexos Multienzimáticos/metabolismo , Esteroide 17-alfa-Hidroxilase/metabolismo , Esteroides/metabolismo , 17-alfa-Hidroxipregnenolona/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Desidroepiandrosterona/metabolismo , Evolução Molecular , Cobaias , Dados de Sequência Molecular , Complexos Multienzimáticos/genética , Mutagênese Sítio-Dirigida , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Esteroide 17-alfa-Hidroxilase/genética , Especificidade por SubstratoRESUMO
The PEA3 subfamily of ETS-domain proteins play important roles in regulating transcriptional activation and have been implicated in several tumorigenic processes. Here we describe the identification of a further member of this family from zebrafish which most likely represents a homologue of PEA3. A high degree of sequence conservation is observed in the ETS DNA-binding domain and acidic transcriptional activation domain. The DNA binding specificity of zebrafish PEA3 is virtually identical to that exhibited by mammalian family members and is autoregulated by cisacting inhibitory domains. Transcriptional activation by zebrafish PEA3 is potentiated by the ERK MAP kinase and protein kinase A pathways. During embryogenesis, PEA3 is expressed in complex spatial and temporal patterns in both mesodermal somites and ectodermal tissues including the brain, dorsal spinal chord and neural crest. Our characterisation of zebrafish PEA3 furthers our understanding of its molecular function and its expression profile suggests a novel role in cell patterning in the early vertebrate embryo.
Assuntos
Proteínas de Ligação a DNA/genética , Quinases de Proteína Quinase Ativadas por Mitógeno , Fatores de Transcrição/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , DNA/metabolismo , DNA Complementar , Proteínas de Ligação a DNA/metabolismo , Humanos , MAP Quinase Quinase 1 , Mamíferos , Camundongos , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/metabolismo , Ativação Transcricional , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-ZebraRESUMO
The Ets family of transcription factors comprises several members which are involved to regulate gene transcription. Although several consensus binding sites for Ets proteins can be found in a wide series of promoter, only a limited number of them are indeed activated by these transcription factors. The human intercellular adhesion molecule-1 (ICAM-1) plays a crucial role in immune responses by enabling the binding of effector cells to various target cell types. ICAM-1 is constitutively expressed at different levels in the absence of stimuli in different cell types, and its expression is upregulated by several proinflammatory cytokines. We have here examined the transcriptional regulation of human ICAM-1 expression by Ets proteins, and more particularly by ERM, a member of this family of transcription factors. Transient transfection assays revealed that Ets-2 and ERM significantly activate the transcription of ICAM-1 promoter, whereas the less-related Ets family member, Spi-1/Pu.1, failed to do so. Transfection of a series of ICAM-1 promoter deletion mutants together with ERM expression plasmids have shown that an Ets responsive element is located within the first 176 bp upstream from the translational start site. Electrophoretic mobility shift assays and DNase I footprinting analysis have enabled us to identify two Ets binding sites at positions -158 and -138 from the ATG, respectively. Site directed mutagenesis of these elements has shown that the distal site is the major element required for the ERM-mediated activation of the ICAM-1 promoter. We can thus conclude that expression of ICAM-1 can be regulated by Ets transcription factors.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Molécula 1 de Adesão Intercelular/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Mapeamento Cromossômico , DNA , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-ets-2 , Proteínas Proto-Oncogênicas/genética , Coelhos , Transativadores/genética , Fatores de Transcrição/genética , Ativação Transcricional , Células Tumorais CultivadasRESUMO
Vascular endothelial cadherin (VE cadherin) gene encodes a Ca2+-dependent cell adhesion molecule required for the organization of interendothelial junctions. This gene is exclusively and constitutively expressed in endothelial cells. Previous data with transgenic mice revealed that the transcriptional regulatory elements present within a -2486/+24 DNA fragment of mouse VE cadherin gene mimic the tissue-specific activity of the endogenous promoter. In this study, we analyzed elements implicated in the function of the proximal regulatory region. Electrophoretic mobility shift assay identified a GT-rich sequence (positions -49/-39) interacting with factors related to the Sp1 family. Point mutations abolished the binding of nuclear proteins in vitro and drastically diminished the activity of the promoter in transient transfection assay. Supershift assays with antibodies against proteins of the Sp1 family revealed that Sp1 and Sp3 interact with this region of the VE cadherin promoter. Furthermore, two GGAA motifs, located at positions -93/-90 and -109/-106, were shown to interact with nuclear factors. Site-directed mutagenesis of these sequences demonstrated that these Ets binding sites are essential for promoter activity. In vitro binding assays in the presence of various antisera suggest that Erg is one of the proteins interacting with the -109/-106 site.
Assuntos
Caderinas/genética , Endotélio Vascular/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Caderinas/metabolismo , Bovinos , Linhagem Celular , DNA , Proteínas de Ligação a DNA/metabolismo , Eletroforese , Endotélio Vascular/citologia , Regulação da Expressão Gênica , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-ets , Fator de Transcrição Sp3 , Células Tumorais CultivadasRESUMO
17 beta-Hydroxysteroid dehydrogenase (17HSD) type 2 efficiently catalyzes the conversion of the high activity 17 beta-hydroxy forms of sex steroids into less potent 17-ketosteroids. In the present study in situ hybridization was utilized to analyze the cellular localization of 17HSD type 2 expression in adult male and female mice. The data indicate that 17HSD type 2 mRNA is expressed in several epithelial cell layers, including both absorptive and secretory epithelia as well as protective epithelium. In both males and females, strong expression of 17HSD type 2 was particularly detected in epithelial cells of the gastrointestinal and urinary tracts. The mRNA was expressed in the stratified squamous epithelium of the esophagus, and surface epithelial cells of the stomach, small intestine and colon. The hepatocytes of the liver and the thick limbs of the loops of Henle in the kidneys, as well as the epithelium of the urinary bladder, also showed strong expression of 17HSD type 2 mRNA in both male and female mice. In the genital tracts, low 17HSD type 2 expression was detected in the seminiferous tubules, the uterine epithelial cells and the surface epithelium of the ovary. Expression of the mRNA was also detected in the sebaceous glands of the skin. The results indicate that in both male and female mice, 17HSD type 2 is expressed mainly in the various epithelial cell types of the gastrointestinal and urinary tracts, and therefore suggest a role for the enzyme in steroid inactivation in a range of tissues and cell types not considered as classical sex steroid target tissues.