RESUMO
X-linked forms of mental retardation (XLMR) include a variety of different disorders and may account for up to 25% of all inherited cases of mental retardation. So far, seven X-chromosomal genes mutated in nonspecific mental retardation (MRX) have been identified: FMR2, GDI1, RPS6KA3, IL1RAPL, TM4SF2, OPHN1 and PAK3 (refs 2-9). The products of the latter two have been implicated in regulation of neural plasticity by controlling the activity of small GTPases of the Rho family. Here we report the identification of a new MRX gene, ARHGEF6 (also known as alphaPIX or Cool-2), encoding a protein with homology to guanine nucleotide exchange factors for Rho GTPases (Rho GEF). Molecular analysis of a reciprocal X/21 translocation in a male with mental retardation showed that this gene in Xq26 was disrupted by the rearrangement. Mutation screening of 119 patients with nonspecific mental retardation revealed a mutation in the first intron of ARHGEF6 (IVS1-11T-->C) in all affected males in a large Dutch family. The mutation resulted in preferential skipping of exon 2, predicting a protein lacking 28 amino acids. ARHGEF6 is the eighth MRX gene identified so far and the third such gene to encode a protein that interacts with Rho GTPases.
Assuntos
Proteínas de Ciclo Celular/genética , Cromossomos Humanos Par 21 , Fatores de Troca do Nucleotídeo Guanina/genética , Deficiência Intelectual/genética , Mutação , Translocação Genética , Cromossomo X , Proteínas rho de Ligação ao GTP/genética , Sequência de Bases , Mapeamento Cromossômico , Feminino , Ligação Genética , Marcadores Genéticos , Humanos , Deficiência Intelectual/enzimologia , Íntrons , Masculino , Dados de Sequência Molecular , Linhagem , Fatores de Troca de Nucleotídeo Guanina RhoRESUMO
Prostaglandin F2 alpha (PGF2 alpha) secretion is lowest at midcycle and highest on day 15 at luteolysis in the cycling guinea pig uterus and is inversely related to serum progesterone levels. An increase in 17-beta estradiol (E2) occurs only towards the end of the cycle. To investigate the effect of steroids on the control of uterine PGF2 alpha metabolism at the level of gene expression we established a primary cell culture model of day 15 cycling guinea pig endometrial cells. We cloned guinea pig cDNAs for cyclooxygenase 2 (COX-2), 15-hydroxyprostaglandin dehydrogenase (PGDH) that converts PGF2 alpha to biologically inactive 13,14-dihydro-15-keto PGF2 alpha (PGFM) and a fragment of cyclooxygenase-1 (COX-1). They were found to bear 87% and 90% homology at the amino acid level to their human counterparts for COX-2 and PGDH, respectively, retaining all functional sites. Purified epithelial and stromal cell subcultures were primed with medium containing either E2 or medroxyprogesterone acetate (MPA) for 24 h. They were then treated for a further 4 or 24 h either withdrawing the steroid, maintaining the priming steroid, or supplementing with both steroids, before harvesting conditioned media and RNA. Epithelial cells secreted 30-fold more PGF2 alpha compared with stromal cells (e.g. 7.8 +/- 0.7 vs. 0.26 +/- 0.09 pg/ng DNA.24 h), and PGF2 alpha secretion levels were approximately 15-fold higher than those of PGFM (e.g. 7.8 +/- 0.7 vs. 0.45 +/- 0.16 pg/ng DNA.24 h, for epithelial cells). COX-1 transcripts were low and unaffected by treatment in both cell types. COX-2 transcripts were more abundant in epithelial than stromal cells. Steroid-modulated, COX-2 dependent changes in PGF2 alpha secretion were observed. The addition of MPA to E2 primed cells caused a decrease in PGF2 alpha secretion and COX-2 messenger RNA levels after 4 h. Conversely, the addition of E2 to MPA primed epithelial cells led to an increase in PGF2 alpha secretion and COX-2 messenger RNA levels after 4 and 24 h. The withdrawal of E2 caused a fall in PGF2 alpha secretion and COX-2 transcripts after 24 h. In contrast, PGDH transcripts were more abundant in stromal than epithelial cells and were up-regulated by the addition of MPA to E2 primed cells. These in vitro observations are in keeping with the secretory profile seen in vivo in the cycling guinea pig uterus suggesting that 1) the fall of E2 and the coinciding rise in progesterone seen in the early cycle lead to a reduction in PGF2 alpha levels; and 2) the rise of E2 in the late cycle on a progesterone primed uterus is the stimulus for an increase in uterine PGF2 alpha production. Our findings suggest a differential role for uterine stroma and epithelium in vivo whereby the former acts to remove (via PGDH), and the latter to produce (via COX-2) biologically active prostaglandin.
Assuntos
Dinoprosta/metabolismo , Endométrio/metabolismo , Hidroxiprostaglandina Desidrogenases/genética , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Ciclo-Oxigenase 2 , Dinoprosta/análogos & derivados , Endométrio/citologia , Feminino , Cobaias , Humanos , Proteínas de Membrana , Dados de Sequência Molecular , RNA Mensageiro/análiseRESUMO
NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) is a key catabolic enzyme in the inactivation of PGF2alpha and PGE2 and therefore serves as an important determinant in regulating their local concentrations. To gain insights into the transcriptional regulation of this enzyme, we have isolated 3.5 kb of the 5'-flanking sequence of the human PGDH promoter and characterized its control in hemopoietic cells and cells of myometrial and placental origin. Several potential binding sites for cAMP-responsive element-binding protein (CREB), Ets, and activating protein-1 (AP-1) transcription factors are present within 2368 bp of the 5'-flanking region. This region and deletions thereof were fused to the luciferase reporter gene and used for transient transfection experiments. In Jurkat leukemic T cells, which express PGDH endogenously, the transfected PGDH promoter was strongly induced by phorbol ester. Induction was reversed by coexpression of A-Fos, a dominant negative to AP-1. In primary cultures of myometrial smooth muscle cells (SMC), the Ets family members Ets-1, Ets-2, and PEA3 potently stimulated transcriptional activity of the PGDH promoter. PEA3-mediated activation was partially repressed by A-Fos, suggesting an involvement of AP-1 proteins, which might be conferred by a distal and a proximal Ets/ AP-1 composite element. The distal Ets/AP-1 element is flanked by two CRE-like sequences. Cotransfection of A-CREB, a dominant negative to CREB, inhibited stimulation of PGDH-2368/luc3 by PEA3 in myometrial SMC, whereas treatment with 8-bromo-cAMP moderately enhanced promoter activity. Progesterone is believed to be an important stimulus for PGDH expression in the utero-placental unit, thus contributing to the maintenance of a quiescent uterus during pregnancy. In myometrial SMC, both isoforms of the progesterone receptor, PR-B and PR-A, caused a ligand-dependent activation of PGDH-2368/luc3. Transcriptional activity of PR-B, but not PR-A, was further enhanced by the addition of 8-bromo-cAMP. We could not confirm a recently proposed transcriptional control of PGDH by mineralocorticoid receptor. No effect of mineralocorticoid receptor, in the absence or presence of aldosterone, with or without 8-bromo-cAMP, was observed on PGDH-2368/luc3. Taken together, these findings demonstrate control of the PGDH promoter by multiple pathways and provide evidence for cross-talk among Ets, AP-1, cAMP, and PR-mediated signaling, suggesting complex regulatory mechanisms for the expression of PGDH.
Assuntos
Regulação Enzimológica da Expressão Gênica , Hidroxiprostaglandina Desidrogenases/genética , Progesterona/fisiologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Coriocarcinoma , Éxons , Feminino , Genes Reporter , Biblioteca Genômica , Células HL-60 , Humanos , Células Jurkat , Luciferases/genética , Camundongos , Dados de Sequência Molecular , Miométrio/enzimologia , Placenta/enzimologia , Gravidez , Proteína Proto-Oncogênica c-ets-1 , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ets , Proteínas Recombinantes de Fusão/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Fator de Transcrição AP-1/genética , Fatores de Transcrição/genética , Transcrição Gênica , Células Tumorais Cultivadas , Neoplasias UterinasRESUMO
A sample of 114 primary breast tumors and corresponding constitutional DNA were tested for loss of heterozygosity (LOH) of the YNZ22 and p53 genes, both located in the 17p13 region. Loss of the p53 allele was found in 28 of 44 primary breast carcinomas (64%). In contrast LOH in only 26 of 61 tumors (43%) was detected with the variable number of tandem repeats (VNTR) probe YNZ22 mapping at 17p13.3 close to the p53 locus at 17p13.1. Among 19 tumors informative for both probes allele loss at 17p13.3 never occurred without p53 involvement. These data suggest, that p53 is the target of 17p13 allelic deletions in human breast cancer. Immunohistochemistry showed overexpression of the p53 protein in 25 of 50 cases (50%) presumably reflecting activating point mutations. Overexpression was not correlated with allele loss but seemed to be closely related to the presence of point mutations in this study. No homozygous deletions or rearrangements of the p53 gene were detected. This would argue for an important role of heterozygous p53 mutations in human breast cancer.
Assuntos
Neoplasias da Mama/genética , Deleção de Genes , Genes p53 , Proteínas de Neoplasias/genética , Proteína Supressora de Tumor p53/genética , Alelos , Sequência de Bases , Neoplasias da Mama/química , Cromossomos Humanos Par 17 , Sondas de DNA , Feminino , Heterozigoto , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Mutação Puntual , Reação em Cadeia da PolimeraseRESUMO
cDNA probe of the casein kinase 2 alpha subunit gene detects a biallelic PstI polymorphism. This restriction fragment length polymorphism is the first known genetic marker of this gene.