RESUMO
CD40 is an important mediator of immune and inflammatory responses. It is a costimulatory molecule for B cell proliferation and survival. Blockade of CD40 has been shown to induce tolerance and its role in other pathogenic conditions has led to the proposal that CD40 inhibition could be valuable therapeutically. As a first step to this end, we have characterized a CD40-dominant negative receptor. This inhibitory mutant lacks the identified CD40 signaling domains. It inhibits both cotransfected and endogenous CD40 activation of NF-kappaB. This mutant is specific, as it does not affect TNF or latent membrane protein 1 signaling. Its potential usefulness is illustrated by its ability to inhibit the CD40 ligand-stimulated increases of HLA and CD54 expression, molecules involved in Ag recognition and lymphocyte recruitment leading to organ rejection. The inhibitory mutant has no TNFR-associated factor 2-binding capabilities and inhibits the recruitment of TNFR-associated factor 2 to the CD40 signaling complex after stimulation. These studies show that the CD40 inhibitory receptor molecule is effective, specific, and useful both for research and potentially as a clinical tool. And furthermore, it is likely that similar dominant inhibitory receptors can be generated for all of the members of the TNFR superfamily.
Assuntos
Antígenos CD40/genética , Antígenos CD40/metabolismo , Deleção de Genes , Genes Dominantes/imunologia , Imunossupressores/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Antígenos CD40/fisiologia , Humanos , Imunofenotipagem , Imunossupressores/farmacologia , Células Jurkat , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Receptores do Fator de Necrose Tumoral/fisiologia , Fator 2 Associado a Receptor de TNF , Transfecção , Células Tumorais CultivadasRESUMO
The latent membrane protein-1 (LMP1) of Epstein-Barr virus induces gene transcription, phenotypic changes, and oncogenic transformation. One cellular gene induced by LMP1 is that for intercellular adhesion molecule-1 (ICAM-1), which participates in a wide range of inflammatory and immune responses. ICAM-1 may enhance the immune recognition of cells transformed by Epstein-Barr virus, and thus combat development of malignancy. Despite growing understanding of the various signaling functions of LMP1, the molecular mechanisms by which LMP1 induces ICAM-1 are not understood. Here, we demonstrate that transcriptional activation by LMP1 is absolutely dependent upon a variant NF-kappaB motif within the tumor necrosis factor alpha (TNFalpha) response element of the ICAM-1 promoter. Although the TNFalpha response element is sufficient for TNFalpha induction of the ICAM-1 promoter, LMP1 also required the cooperation of additional upstream sequences for optimal induction. Inhibitor studies of known LMP1-induced signaling pathways ruled out the involvement of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase, and the Janus-activating tyrosine kinase 3 (JAK3), and confirmed NF-kappaB as a critical factor for induction of ICAM-1. However, although constitutive activation of NF-kappaB efficiently induced promoter activity, it was not sufficient to induce either ICAM-1 mRNA or ICAM-1 protein. Using signaling defective LMP1 mutants and deacetylation inhibitors, we showed that the C-terminal activator region 1 of LMP1 delivers a new cooperating signal to induce ICAM-1 mRNA.
Assuntos
Herpesvirus Humano 4/genética , Proteínas I-kappa B , Molécula 1 de Adesão Intercelular/genética , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Transcrição Gênica , Ativação Transcricional , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Linfócitos B , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Genes Reporter , Humanos , Imidazóis/farmacologia , Molécula 1 de Adesão Intercelular/análise , Células Jurkat , Luciferases/genética , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidor de NF-kappaB alfa , Proteínas Oncogênicas Virais/metabolismo , Piridinas/farmacologia , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossíntese , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transfecção , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Two genes encoding the latent membrane protein 1 (LMP1) of the Epstein-Barr virus (EBV) were isolated from a single case of Hodgkin's disease (HD) and were tested for their biological activities. The LMP1 gene from the Reed-Sternberg cells contained point mutations relative to the prototype LMP1 gene, leading to amino-acid exchanges. The LMP1 gene from passenger lymphocytes showed identical point mutations, but also had an in-frame insertion of 132 base pairs within the 33-bp repeat region. This insert encoding 44 amino acids contained the sequence PSQQS, corresponding to the potential TRAF-binding motif PXQXT/S. When compared to the B95.8 gene, both HD-derived LMP1 genes showed an increase in the transformation of Rat-1 rodent fibroblasts. The transforming ability of the LMP1 gene with the insertion was greater than that of the other HD-derived LMP1, and was comparable with the highly transforming LMP1-Cao gene derived from a nasopharyngeal carcinoma. The HD-derived genes stimulated expression of the cell-surface markers, CD40 and CD54, similarly to the LMP1-B95.8 gene, while the LMP1-Cao gene had a significantly reduced ability to induce these proteins. In contrast, the LMP1-Cao transactivated an NF-kappaB-response element more efficiently than did the HD-derived genes. Transfer of the 132-bp insert alone into the B95.8 gene did not increase its transforming activity to the LMP1-Cao level, indicating that additional mutations in the LMP1 gene are necessary for modulating this function.
