RESUMEN
Patients with sickle cell disease exhibit both acute and chronic activation of the coagulation and fibrinolytic systems. To test the relationship between sickle cell pathology and activation of the hemostatic system, mice with targeted deletions of plasminogen (Plg) or fibrinogen (Fib) were crossed with transgenic mice expressing Hb SAD [beta(6Glu-Val) (HbS), beta(23Val-Ile) (HbAntilles), and beta(121Glu-Gln) (HbD-Punjab)]. Fibrinogen deficiency dramatically reduced the survival of mice with Hb SAD to a much greater degree than mice with normal hemoglobin. The combination of Hb SAD and fibrinogen deficiency had a greater effect on mortality than that obtained by adding the mortality risks of each defect alone. The deleterious effect of the combination of Hb SAD and fibrinogen deficiency on mortality was accelerated by hypoxia. The excess mortality associated with plasminogen deficiency was identical in SAD and control mice. The adverse effect of fibrinogen deficiency on mortality in SAD mice is not consistent with the simple hypothesis that fibrin deposition is uniformly deleterious in the context of vaso-occlusive sickle cell disease. Rather, our findings suggest that the contribution of fibrinogen to tissue repair may in some contexts limit sickle cell disease pathophysiology.
Asunto(s)
Anemia de Células Falciformes/genética , Fibrinógeno/genética , Hemoglobina Falciforme/genética , Plasminógeno/deficiencia , Anemia de Células Falciformes/mortalidad , Animales , Eliminación de Gen , Genotipo , Ratones , Ratones Transgénicos , Plasminógeno/genética , Análisis de SupervivenciaRESUMEN
OBJECTIVE: The KCl cotransporter (KCC) plays an important role in cellular cation and volume regulation and contributes to the process of volume reduction that accompanies reticulocyte maturation. In human red cells containing sickle hemoglobin, KCl cotransporter activity is high compared to normal cells, and contributes to the deleterious dehydration of sickle reticulocytes. To date, genes for four KCC isoforms have been identified. As a step toward determining which isoform(s) is responsible for the Cl-dependent K fluxes in reticulocytes, human erythroid cells were examined for the presence of various KCC isoform transcripts. METHODS: In vitro differentiated erythroid precursors, and reticulocytes isolated from normal individuals and sickle patients, were examined by reverse-transcriptase PCR for the expression of KCC isoforms. Transient transfection experiments were subsequently performed to characterize a novel KCC1 promoter. RESULTS: Expression of multiple isoforms was detected, with transcripts for KCC1, 3, and 4 detected in all samples of erythroid cells. Two N-terminal splicing variants were detected for both KCC1 and 3. Sickle hemoglobin containing reticulocytes demonstrated KCC isoform expression patterns similar to wild-type cells, except for a consistent difference in the relative abundance of one KCC1 splice variant. This N-terminal variant initiates from a newly described promoter in the KCC1 gene. CONCLUSION: Three KCC genes are expressed in human red cells. Splicing variants arising from the KCC1 and 3 genes are also evident. Structure/function studies of mouse KCC1 suggest that these natural variants could profoundly affect overall cotransporter activity in the red cell.
Asunto(s)
Anemia de Células Falciformes/patología , Eritrocitos/metabolismo , Isoformas de Proteínas/metabolismo , Simportadores/metabolismo , Anemia de Células Falciformes/inmunología , Células Cultivadas , Humanos , Isoformas de Proteínas/genética , Empalme del ARN , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Simportadores/genética , Cotransportadores de K ClRESUMEN
Ectopic expression of LMO2 occurs in approximately 45% of T-lineage acute lymphoblastic leukemias (T-ALL), sometimes in association with chromosomal translocations. Recently, a lymphoproliferative disorder developed in two participants in a gene therapy trial due to LMO2 activation via integration of the retroviral vector. To investigate these regulatory disruptions, we analyzed the promoter region and identified a tissue-specific repressor. The fragment containing this element could also produce tissue-specific suppression of transcription from the SV40 promoter. This suppression involves histone acetylation which can be relieved with Trichostatin A (TSA). The negative element is in a region consistently removed from LMO2 in the known chromosomal translocations.
Asunto(s)
Proteínas de Unión al ADN/genética , Leucemia/genética , Metaloproteínas/genética , Proto-Oncogenes , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción Genética , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Bases , Secuencia de Consenso , Histona Desacetilasas/fisiología , Humanos , Ácidos Hidroxámicos/farmacología , Células Jurkat , Células K562 , Proteínas con Dominio LIM , Datos de Secuencia Molecular , Especificidad de Órganos , Proteínas Proto-Oncogénicas , TransfecciónRESUMEN
Most K-Cl cotransport in the erythrocyte is attributed to potassium chloride cotransporter 1 (KCC1). K-Cl cotransport is elevated in sickle erythrocytes, and the KCC1 gene has been proposed as a modifier gene in sickle cell disease. To provide insight into our understanding of the regulation of the human KCC1 gene, we mapped the 5' end of the KCC1 cDNA, cloned the corresponding genomic DNA, and identified the KCC1 gene promoter. The core promoter lacks a TATA box and is composed of an initiator element (InR) and a downstream promoter element (DPE), a combination found primarily in Drosophila gene promoters and rarely observed in mammalian gene promoters. Mutational analyses demonstrated that both the InR and DPE sites were critical for full promoter activity. In vitro DNase I footprinting, electrophoretic mobility shift assays, and reporter gene assays identified functional AP-2 and Sp1 sites in this region. The KCC1 promoter was transactivated by forced expression of AP-2 in heterologous cells. Sequences encoding the InR, DPE, AP-2, and Sp1 sites were 100% conserved between human and murine KCC1 genes. In vivo studies using chromatin immunoprecipitation assays with antihistone H3 and antihistone H4 antibodies demonstrated hyperacetylation of this core promoter region.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas/genética , Simportadores/genética , Simportadores/metabolismo , Factores de Transcripción/metabolismo , Regiones no Traducidas 5'/genética , Acetilación , Secuencia de Bases , Carcinoma Hepatocelular , Cromatina , Clonación Molecular , Células Eritroides , Células HeLa , Humanos , Células K562 , Datos de Secuencia Molecular , Pruebas de Precipitina , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción AP-2 , Sitio de Iniciación de la Transcripción , Cotransportadores de K ClRESUMEN
Six to eight copies of a transgene integrated into mouse chromosome 15 resulting in a new transgene insertional mutant, Footless, presenting with malformations of the limbs, kidney, and soft palate. Homozygotes possess a unique asymmetric pattern of limb truncations. Posterior structures from the autopod and zeugopod of the hindlimbs are missing with left usually more severely affected than right. In contrast, anterior structures are missing from the right forelimbs. The left forelimb is usually normal except for the absence of the distal telephalanges and nails. These structures are absent on all formed digits. In situ hybridization assays examined the expression of Shh, dHand, Msx2, Fgf8, En1, and Lmx1b in mutant limb buds and indicated normal establishment of the anterior/posterior and dorsal/ventral axes of the developing limbs. However, dysmorphology of the apical ectodermal ridge was observed in the mutant limb buds.
Asunto(s)
Extremidades/embriología , Deformidades Congénitas de las Extremidades/genética , Animales , Southern Blotting , Fisura del Paladar/embriología , Proteínas de Unión al ADN/metabolismo , Femenino , Factor 8 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/fisiología , Genotipo , Heterocigoto , Proteínas de Homeodominio , Homocigoto , Hibridación in Situ , Riñón/embriología , Ratones , Modelos Genéticos , Mutación , Paladar Blando/embriología , Fenotipo , Factores de Tiempo , TransgenesRESUMEN
The transcription factor LMO2 is believed to exert its effect through the formation of protein-protein interactions with other DNA-binding factors such as GATA-1 and TAL1. Although LMO2 has been shown to be critical for the formation of the erythroid cell lineage, the gene is also expressed in a number of nonerythroid tissues. In this report, we demonstrate that the more distal of the 2 promoters for the LMO2 gene is highly restricted in its pattern of expression, directing the hematopoietic-specific expression of this gene. Deletion and mutation analyses have identified a critical cis element in the first untranslated exon of the gene. This element is a consensus-binding site for a small family of basic leucine zipper proteins containing a proline and acidic amino acid-rich (PAR) domain. Although all 3 members of this family are produced in erythroid cells, only 2 of these proteins, thyrotroph embryonic factor and hepatic leukemia factor, can activate transcription from this LMO2 promoter element. These findings represent a novel mechanism in erythroid gene regulation because PAR proteins have not previously been implicated in this process.