RESUMO
We have cloned the rat homologue of the ring-H2 protein Goliath involved in Drosophila development. The rat Goliath mRNA (1.85 kb) was translated as a major ubiquitous protein species of 28-kDa and three larger isoforms (50, 46, and 36 kDa) expressed mainly in liver, lung, stomach, heart, and thymus and barely detectable in other tissues (kidney, skeletal muscle, brain, testis, intestine, and spleen). By immunohistochemistry on rat testis sections, we localized the protein in interstitial tissue and seminiferous tubules. In tubules, Goliath was expressed mainly in postmeiotic germ cells and to a much lesser extent in Sertoli cells. In the interstitium, Goliath was exclusively present in Leydig cells. Using a series of immunolabeling, cellular fractionation, and electron microscopy experiments, we established that Goliath is present in mitochondria of the R2C Leydig cell line. Using short-term hypophysectomized animals, we showed that Goliath is regulated by LH/hCG in Leydig cells but not in germ cells. This regulation in Leydig cells concerned only the 50-kDa isoform. This report is the first description of a differential regulation of the Goliath protein between germ cells and Leydig cells.
Assuntos
Gonadotropina Coriônica/metabolismo , Células Intersticiais do Testículo/fisiologia , Hormônio Luteinizante/metabolismo , Proteínas Mitocondriais/genética , Animais , Linhagem Celular , Clonagem Molecular , DNA Complementar , Regulação da Expressão Gênica/fisiologia , Hipofisectomia , Imuno-Histoquímica , Células Intersticiais do Testículo/citologia , Masculino , Proteínas Mitocondriais/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Espermátides/citologia , Espermátides/fisiologia , Testículo/citologia , Dedos de Zinco/genéticaRESUMO
By systematic analysis of a human testis library, we have isolated the hH-Rev107-3 cDNA, identical to hH-Rev107-1 cDNA, which was previously described as a class II tumor suppressor gene. In this study, two transcripts (1 and 0.8 kb) were detected by Northern blot in all human tissues, excepted in thymus. The strongest expression was found in testis, skeletal muscle and heart. These two mRNA are probably transcribed from only one gene that we mapped to the q12-q13 region of the chromosome 11. In human testis, hH-Rev107 gene expression was localized, by in situ hybridization, within the round spermatids. To investigate a possible role for hH-Rev107 protein in testicular malignant growth, we examined the expression of this gene in germ cell tumors. A strong hH-Rev107 gene expression was observed in normal testis as well as in samples with preinvasive carcinoma in situ but was completely absent in overt tumors, both seminomas and non-seminomas. By in situ hybridization, CIS was found hH-Rev107 positive and tumor negative. A semi-quantitative assessment of hH-Rev107 mRNA level in testicular germ cell tumors, by RT-PCR, exhibited a ninefold decrease in the gene expression. No gross structural aberrations of hH-Rev107 gene were detected in these human primary tumors. The results suggest that down-regulation of hH-Rev107 may be associated with invasive progression of testicular germ cell tumors.
Assuntos
Meiose , Neoplasias Embrionárias de Células Germinativas/metabolismo , Biossíntese de Proteínas , Proteínas/genética , Espermatozoides/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Mapeamento Cromossômico , Cromossomos Humanos Par 11 , Regulação para Baixo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Dados de Sequência Molecular , Fosfolipases A2 Independentes de Cálcio , Testículo/metabolismo , Distribuição Tecidual , Proteínas Supressoras de TumorRESUMO
When ectopically expressed, the serine/threonine kinase Mos can induce oncogenic transformation of somatic cells by direct phosphorylation of MAP kinase/ERK kinase (MEK1), activating the mitogen-activated protein kinases ERK1 and ERK2. On the other hand, overexpression of Mos in C2C12 myoblasts is not transforming. Mos activates myogenic differentiation by promoting heterodimerization of the MyoD/E12 proteins, increasing the expression of myogenic markers and the positive autoregulatory loop of MyoD. In this study, we show that in myogenic cells, the mitogenic and oncogenic signalling from the Mos/MEK/ERK pathway is suppressed by MyoD through the formation of a heterotrimeric complex.
Assuntos
Sistema de Sinalização das MAP Quinases , Proteína MyoD/metabolismo , Proteínas Oncogênicas v-mos/metabolismo , Proteínas Serina-Treonina Quinases , Animais , Western Blotting , Diferenciação Celular , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase/metabolismo , Luciferases/metabolismo , MAP Quinase Quinase 1 , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteína MyoD/genética , Proteínas Oncogênicas v-mos/genética , Fosforilação , Plasmídeos/metabolismo , Ligação Proteica , Transdução de Sinais , TransfecçãoRESUMO
The myogenic b-HLH transcription factor MyoD activates expression of muscle-specific genes and autoregulates positively its own expression. Various factors such as growth factors and oncogene products repress transcriptional activity of MyoD. The c-mos proto-oncogene product, Mos, is a serine/threonine kinase that can activate myogenic differentiation by specific phosphorylation of MyoD which favors heterodimerization of MyoD and E12 proteins. Here we show that overexpression of Mos enhances the expression level of MyoD protein in myoblasts although phosphorylation of MyoD by Mos does not modify its stability but promotes transcriptional transactivation of the MyoD promoter linked to the luciferase reporter gene. Moreover, co-expression of MyoD with Mos(wt) but not with the kinase-inactive Mos(KM) greatly enhances expression of endogenous MyoD protein and the DNA binding activity of MyoD/E12 heterodimers in 10T1/2 cells. Our data suggest that Mos increases the ability of MyoD to transactivate both muscle-specific genes and its own promoter and could therefore participate in the positive autoregulation loop of MyoD and muscle differentiation.
Assuntos
Proteína MyoD/metabolismo , Proteínas Proto-Oncogênicas c-mos/metabolismo , Regulação para Cima , Animais , Linhagem Celular , DNA/metabolismo , Camundongos , Músculo Esquelético , Proteína MyoD/genética , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
We have cloned and sequenced an almost complete c-DNA and the entire genomic sequence of rat the H19 gene, which is developmentally regulated in skeletal muscle. The data base comparison revealed a 92% homology with mouse gene H19. However the rat H19 ORFs do not display significant homology with the H19 ORFs from other species. In contrast to the mouse, the rat H19 mRNA is not easily detectable in fetal rat skeletal fibers. Its level increases after birth (up to 12 to 18 days) and remains stable thereafter. The pattern of H19 mRNA expression in rat muscle in vivo is very similar to the c-mos gene expression in this tissue, suggesting an interrelationship between H19 and c-mos products during muscle differentiation. Indeed, our results indicate that overexpression of c-mos protein in the muscle cell line C2C12 induces a concomitant increase of H19 mRNA expression. Furthermore, repression of c-mos protein expression by anti-sense RNAs extinguishes H19 mRNA expression and inhibits the differentiation process. These data suggest a relationship between c-mos and H19 expression and, in addition, the involvement of both gene products in the process of myogenesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Genes Supressores de Tumor , Genes mos , Músculo Esquelético/embriologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Diferenciação Celular/genética , Linhagem Celular , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta , RNA Antissenso/farmacologia , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência do Ácido NucleicoRESUMO
A rat adult skeletal muscle probe (Asm15) originated from a rhabdomyosarcoma was used to isolate the human homologous sequence from a placenta cDNA library. Among several positive clones the longest EcoRI-EcoRI insert (ASM1) obtained was 1875 bp long with 72% homology with rat Asm15 cDNA sequence. Important variations of ASM1 RNA level were observed in different adult skeletal muscles. Expression of a 29kD ASM1 protein was demonstrated in human adult skeletal muscle lysates using an antiserum (PB1579) raised against the C terminal region of the rat Asm15 protein. The human ASM gene was assigned by somatic cell analysis with human (ASM1) and rat (Asm15) probes to chromosome 11, and by in situ hybridization with the human probe to 11p15, a chromosome region involved in human embryonal rhabdomyosarcomas. Except for the presence of a HindII restriction site, the results obtained for the restriction map and the sequence of ASM1 cDNA (data not shown) exhibited extensive homology with the human H19 DNA sequence which have been mapped with a mouse probe also in 11p15. This suggests that ASM/Asm and H19 may represent the same sequence (in this hypothesis the presence of the supplementary HindII site in our ASM1 probe is explained by polymorphic variability). However it was reported that human and mouse H19 mRNA did not encode for a protein but acted as an RNA molecule whereas in our present study ASM protein was detected in human adult skeletal muscle. This could be explained by important regulation of ASM protein expression during development and cell differentiation. However we cannot exclude for the different species studied (mouse, rat, and man) the hypothesis that H19 and ASM/Asm mRNA may represent two distinct messengers from the same gene or even from duplicated genes.
Assuntos
Cromossomos Humanos Par 11 , Músculos/fisiologia , Adulto , Animais , Southern Blotting , Linhagem Celular , Bandeamento Cromossômico , Mapeamento Cromossômico , DNA/genética , DNA/isolamento & purificação , Sondas de DNA , Feminino , Humanos , Camundongos , Placenta/fisiologia , Gravidez , Ratos , Mapeamento por Restrição , Rabdomiossarcoma/genéticaRESUMO
The localization of two enkephalin-hydrolysing aminopeptidases i.e. aminopeptidase M (aminopeptidase N, EC 3.4.11.2) relatively insensitive to puromycin (Ki = 78 microM), and a puromycin-sensitive aminopeptidase (Ki = 1 microM) was studied in rat brain. The two aminopeptidases were differentially identified and/or localized using polyclonal anti-aminopeptidase M antibodies displaying anticatalytic activity and the inhibitors puromycin, bestatin and amastatin. Microvessels represent a major localization of cerebral aminopeptidase M as shown by the intense immunostaining of their walls in sections from various regions as well as in a fraction isolated from cerebral cortex homogenates by a sieving procedure. As compared to the starting homogenate, aminopeptidase M activity was enriched about twenty fold in this microvascular fraction. Aminopeptidase M was identified in this fraction by comparing the inhibitory potencies of antibodies and peptidase inhibitors towards the hydrolysis of [tyrosyl-3,5-3H, Met5]enkephalin to those found for the purified enzyme. A rather high aminopeptidase M activity was also localized in choroid plexuses. Following differential and gradient centrifugation analysis of cerebral cortex homogenates, aminopeptidase M activity was also enriched (by five to six fold) in fractions containing synaptic membranes. No significant soluble aminopeptidase M activity could be detected. These data suggest a dual localization of cerebral aminopeptidase M in microvessels and synaptic membranes consistent with its roles in preventing the access of circulating peptides to brain as well as in inactivating neuropeptides released from cerebral neurones. In comparison, puromycin-sensitive aminopeptidase activity, which is about 100 fold higher than aminopeptidase M activity in brain, was relatively low in microvessels and non-detectable in fractions enriched in synaptic membranes, being almost entirely restricted to soluble fractions.
Assuntos
Aminopeptidases/metabolismo , Córtex Cerebral/irrigação sanguínea , Músculo Liso Vascular/enzimologia , Frações Subcelulares/enzimologia , Animais , Antígenos CD13 , Técnicas Imunoenzimáticas , Masculino , Microcirculação/enzimologia , Ratos , Ratos Endogâmicos , Membranas Sinápticas/enzimologiaRESUMO
In addition to "enkephalinase" (EC 3.4.24.11), two enkephalin-hydrolyzing aminopeptidases recently identified in cerebral membranes--aminopeptidase M (EC 3.4.11.2) and a "puromycin-sensitive" aminopeptidase (also designated "MII" or "aminoenkephalinase")--are potentially involved in endogenous enkephalin inactivation. Their participation in the hydrolysis of the endogenous (Met5)enkephalin released by depolarization of slices from rat globus pallidus was assessed, using three inhibitory agents: bestatin, puromycin, and anti-aminopeptidase M antibodies. The selectivity and potency of these agents were first determined by evaluating their IC50 values for inhibition of [3H](Met5)enkephalin hydrolysis by increasingly complex preparations comprising semipurified aminopeptidases, pallidal membranes, and pallidal slices. Bestatin was a fairly potent inhibitor but lacked selectivity, as there was only a 3-fold difference between its IC50 values for the two aminopeptidases, and it displayed restricted diffusion and degradation in the slice preparation. Puromycin discriminated well between the two aminopeptidases (30-fold difference in IC50 values) and did not show any apparent restricted diffusion in the slice preparation. Antiaminopeptidase M antibodies were highly discriminant (greater than 300-fold difference in IC50 values for the two aminopeptidases) but displayed restricted diffusion. Analysis of the concentration-protection curves of the three agents for recovery of the (Met5)enkephalin released from pallidal slices in the presence of the "enkephalinase" inhibitor, thiorphan, indicated that both aminopeptidases participated in enkephalin degradation but that the role of aminopeptidase M was largely predominant, in contrast with its low relative activity in the preparation.
Assuntos
Aminopeptidases/fisiologia , Encéfalo/enzimologia , Encefalina Metionina/metabolismo , Leucina/análogos & derivados , Puromicina/farmacologia , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/imunologia , Animais , Anticorpos/imunologia , Antígenos CD13 , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Globo Pálido/enzimologia , Hidrólise , Técnicas In Vitro , Cinética , Leucina/farmacologia , Masculino , Ratos , Ratos EndogâmicosRESUMO
A predominantly microvascular localization of aminopeptidase M (APM) was established both by immunohistochemistry and biochemical studies of isolated microvessels. In addition a high relative specific activity of APM was evidenced in fractions enriched in synaptic membranes.