RESUMO
ASC/PYCARD is a common adaptor for a diverse set of inflammasomes that activate caspase-1, most prominently the NLR-based inflammasome. Mounting evidence indicates that ASC and these NLRs also elicit non-overlapping functions, but the molecular basis for this difference is unclear. To address this, we performed microarray and network analysis of ASC shRNA knockdown cells. In pathogen-infected cells, an ASC-dependent interactome is centered on the mitogen-activated protein kinase (MAPK) ERK and on multiple chemokines. ASC did not affect the expression of MAPK but affected its phosphorylation by pathogens and Toll-like receptor agonists via suppression of the dual-specificity phosphatase, DUSP10/MKP5. Chemokine induction, DUSP function, and MAPK phosphorylation were independent of caspase-1 and IL-1ß. MAPK activation by pathogen was abrogated in Asc(-/-) but not Nlrp3(-/-), Nlrc4(-/-), or Casp1(-/-) macrophages. These results demonstrate a function for ASC that is distinct from the inflammasome in modulating MAPK activity and chemokine expression and further identify DUSP10 as a novel ASC target.
Assuntos
Quimiocinas/biossíntese , Proteínas do Citoesqueleto/metabolismo , Fosfatases de Especificidade Dupla/metabolismo , Inflamassomos/metabolismo , Macrófagos/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Proteínas Adaptadoras de Sinalização CARD , Linhagem Celular , Quimiocinas/genética , Proteínas do Citoesqueleto/genética , Fosfatases de Especificidade Dupla/genética , Ativação Enzimática/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Inflamassomos/genética , Macrófagos/citologia , Camundongos , Camundongos Knockout , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Fosfatases da Proteína Quinase Ativada por Mitógeno/genéticaRESUMO
Periodontal disease is a chronic inflammatory disorder that leads to the destruction of tooth-supporting tissue and affects 10-20 million people in the U.S. alone. The oral pathogen Porphyromonas gingivalis causes inflammatory host response leading to periodontal and other secondary inflammatory diseases. To identify molecular components that control host response to P. gingivalis in humans, roles for the NLR (NBD-LRR) protein, NLRP3 (cryopyrin, NALP3), and its adaptor apoptotic speck protein containing a C-terminal caspase recruitment domain (ASC) were studied. P. gingivalis strain A7436 induces cell death in THP1 monocytic cells and in human primary peripheral blood macrophages. This process is ASC and NLRP3 dependent and can be replicated by P. gingivalis LPS and Escherichia coli. P. gingivalis-induced cell death is caspase and IL-1 independent and exhibits morphological features consistent with necrosis including loss of membrane integrity and release of cellular content. Intriguingly, P. gingivalis-induced cell death is accompanied by the formation of ASC aggregation specks, a process not previously described during microbial infection. ASC specks are observed in P. gingivalis-infected primary human mononuclear cells and are dependent on NLRP3. This work shows that P. gingivalis causes ASC- and NLRP3-dependent necrosis, accompanied by ASC speck formation.
Assuntos
Infecções por Bacteroidaceae/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Macrófagos/microbiologia , Monócitos/microbiologia , Necrose/metabolismo , Infecções por Bacteroidaceae/imunologia , Infecções por Bacteroidaceae/patologia , Western Blotting , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/imunologia , Proteínas do Citoesqueleto/imunologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Microscopia Eletrônica de Transmissão , Monócitos/imunologia , Monócitos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Necrose/imunologia , Necrose/microbiologia , Porphyromonas gingivalis , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The Epstein-Barr virus (EBV) BMRF1 gene encodes an early lytic protein that functions not only as the viral DNA polymerase processivity factor but also as a transcriptional activator. BMRF1 has been previously shown to activate transcription of an EBV early promoter, BHLF1, though a GC-rich motif which binds to SP1 and ZBP-89, although the exact mechanism for this effect is not known (D. J. Law, S. A. Tarle, and J. L. Merchant, Mamm. Genome 9:165-167, 1998). Here we demonstrate that BMRF1 activates transcription of the cellular gastrin gene in telomerase-immortalized keratinocytes. Furthermore, BMRF1 activated a reporter gene construct driven by the gastrin promoter in a variety of cell types, and this effect was mediated by two SP1/ZBP-89 binding sites in the gastrin promoter. ZBP-89 has been previously shown to negatively regulate the gastrin promoter. However, ZBP-89 can function as either a negative or positive regulator of transcription, depending upon the promoter and perhaps other, as-yet-unidentified factors. BMRF1 increased the binding of ZBP-89 to the gastrin promoter, and a ZBP-89-GAL4 fusion protein was converted into a positive transcriptional regulator by cotransfection with BMRF1. BMRF1 also enhanced the transcriptional activity of an SP1-GAL4 fusion protein. These results suggest that BMRF1 activates target promoters through its effect on both the SP1 and ZBP-89 transcription factors. Furthermore, as the EBV genome is present in up to 10% of gastric cancers, and the different forms of gastrin are growth factors for gastrointestinal epithelium, our results suggest a mechanism by which lytic EBV infection could promote the growth of gastric cells.
