RESUMEN
A disintegrin and metalloproteinase 10 (ADAM10) is a zinc-dependent proteinase related to matrix metalloproteinases. ADAM10 has emerged as a key regulator of cellular processes by cleaving and shedding extracellular domains of multiple transmembrane receptors and ligands. We have developed B cell-specific ADAM10-deficient mice (ADAM10(B-/-)). In this study, we show that ADAM10 levels are significantly enhanced on germinal center B cells. Moreover, ADAM10(B-/-) mice had severely diminished primary and secondary responses after T-dependent immunization. ADAM10(B-/-) displayed impaired germinal center formation, had fewer follicular Th cells, decreased follicular dendritic cell networks, and altered chemokine expression in draining lymph nodes (LNs). Interestingly, when spleen and LN structures from immunized mice were analyzed for B and T cell localization, tissues structure was aberrant in ADAM10(B-/-) mice. Importantly, when ADAM10-deficient B cells were stimulated in vitro, they produced comparable Ab as wild type B cells. This result demonstrates that the defects in humoral responses in vivo result from inadequate B cell activation, likely because of the decrease in follicular Th cells and the changes in structure. Thus, ADAM10 is essential for the maintenance of lymphoid structure after Ag challenge.
Asunto(s)
Proteínas ADAM/fisiología , Secretasas de la Proteína Precursora del Amiloide/fisiología , Inmunidad Humoral , Proteínas de la Membrana/fisiología , Proteínas ADAM/biosíntesis , Proteínas ADAM/deficiencia , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/biosíntesis , Secretasas de la Proteína Precursora del Amiloide/deficiencia , Animales , Subgrupos de Linfocitos B/enzimología , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/patología , Células CHO , Cricetinae , Centro Germinal/enzimología , Centro Germinal/inmunología , Centro Germinal/patología , Inmunidad Humoral/genética , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/deficiencia , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Ganglios Linfáticos Agregados/enzimología , Ganglios Linfáticos Agregados/inmunología , Ganglios Linfáticos Agregados/patología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/enzimología , Subgrupos de Linfocitos T/inmunología , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
Diverse functions, including DNA replication, recombination and repair, occur during S phase of the eukaryotic cell cycle. It has been proposed that p53 and BLM help regulate these functions. We show that p53 and BLM accumulated after hydroxyurea (HU) treatment, and physically associated and co-localized with each other and with RAD51 at sites of stalled DNA replication forks. HU-induced relocalization of BLM to RAD51 foci was p53 independent. However, BLM was required for efficient localization of either wild-type or mutated (Ser15Ala) p53 to these foci and for physical association of p53 with RAD51. Loss of BLM and p53 function synergistically enhanced homologous recombination frequency, indicating that they mediated the process by complementary pathways. Loss of p53 further enhanced the rate of spontaneous sister chromatid exchange (SCE) in Bloom syndrome (BS) cells, but not in their BLM-corrected counterpart, indicating that involvement of p53 in regulating spontaneous SCE is BLM dependent. These results indicate that p53 and BLM functionally interact during resolution of stalled DNA replication forks and provide insight into the mechanism of genomic fidelity maintenance by these nuclear proteins.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Adenosina Trifosfatasas/metabolismo , ADN Helicasas/metabolismo , Replicación del ADN , Recombinación Genética , Proteína p53 Supresora de Tumor/metabolismo , Adenosina Trifosfatasas/genética , Síndrome de Bloom , Bromodesoxiuridina/metabolismo , Ciclo Celular/fisiología , Línea Celular , ADN Helicasas/genética , Proteínas de Unión al ADN/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Hidroxiurea/metabolismo , Modelos Genéticos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Unión Proteica , Recombinasa Rad51 , RecQ Helicasas , Serina/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Hepatitis B virus (HBV) is a major risk factor for hepatocellular carcinoma (HCC). HBV encodes an oncogenic HBx gene that functions as a transcriptional coactivator of multiple cellular genes. To understand the role(s) of HBx in the early genesis of HCC, we systematically analyzed gene expression profiles by serial analysis of gene expression (SAGE) in freshly isolated human primary hepatocytes infected with a replication-defective adenovirus containing HBx. A total of 19,501 sequence tags (representing 1443 unique transcripts) were analyzed, which provide a distribution of a transcriptome characteristic of normal hepatocytes and a profile associated with HBx expression. Examples of the targeted genes were confirmed by the Megarray analysis with a significant correlation between quantitative SAGE and Megarray (r = 0.8, P < 0.005). In HBx-expressing hepatocytes, a total of 57 transcripts (3.9%) were induced, and 46 transcripts (3.3%) were repressed by more than fivefold. Interestingly, most of the HBx-up-regulated transcripts can be clustered into three major classes, including genes that encode ribosomal proteins, transcription factors with zinc-finger motifs, and proteins associated with protein degradation pathways. These results suggest that HBx may function as a major regulator in common cellular pathways that, in turn, regulate protein synthesis, gene transcription, and protein degradation.