Protein kinase Calpha is involved in interferon regulatory factor 3 activation and type I interferon-beta synthesis.
Protein kinase C (PKC) isoforms are critically involved in the regulation of innate immune responses. Herein, we investigated the role of conventional PKCalpha in the regulation of IFN-beta gene expression mediated by the Toll-like receptor 3 (TLR3) signaling pathway. Inhibition of conventional PKC (cPKC) activity in monocyte-derived dendritic cells or TLR3-expressing cells by an isoform-specific inhibitor, Gö6976, selectively inhibited IFN-beta synthesis induced by double-stranded RNA polyinosine-polycytidylic acid. Furthermore, reporter gene assays confirmed that PKCalpha regulates IFN-beta promoter activity, since overexpression of dominant negative PKCalpha but not PKCbeta(I) repressed interferon regulatory factor 3 (IRF-3)-dependent but not NF-kappaB-mediated promoter activity upon TLR3 engagement in HEK 293 cells. Dominant negative PKCalpha inhibited IRF-3 transcriptional activity mediated by overexpression of TIR domain-containing adapter inducing IFN-beta and Tank-binding kinase-1. Additional biochemical analysis demonstrated that Gö6976-treated dendritic cells exhibited IRF-3 phosphorylation, dimerization, nuclear translocation, and DNA binding activity analogous to their control counterparts in response to polyinosine-polycytidylic acid. In contrast, co-immunoprecipitation experiments revealed that TLR3-induced cPKC activity is essential for mediating the interaction of IRF-3 but not p65/RelA with the co-activator CREB-binding protein. Furthermore, PKCalpha knock-down with specific small interfering RNA inhibited IFN-beta expression and down-regulated IRF-3-dependent promoter activity, establishing PKCalpha as a component of TLR3 signaling that regulates IFN-beta gene expression by targeting IRF-3-CREB-binding protein interaction. Finally, we analyzed the involvement of cPKCs in other signaling pathways leading to IFN-beta synthesis. These experiments revealed that cPKCs play a role in the synthesis of IFN-beta induced via both TLR-dependent and -independent pathways.
Johnson
Jolyn
J
Institute for Medical Immunology, Université Libre de Bruxelles, 8 Rue Adrienne Bolland, 6041 Charleroi, Belgium.
Albarani
Valentina
V
Nguyen
Muriel
M
Goldman
Michel
M
Willems
Fabienne
F
Aksoy
Ezra
E
D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000502:physiology; D002227:Carbazoles / Q000494:pharmacology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D050838:Interferon Regulatory Factor-3 / Q000235:genetics / Q000378:metabolism*; D016899:Interferon-beta / Q000096:biosynthesis* / Q000235:genetics; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011070:Poly I-C / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D051196:Toll-Like Receptor 3 / Q000378:metabolism*; D051996:Transcription Factor RelA / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology
D002227:Carbazoles; D004791:Enzyme Inhibitors; C494232:IRF3 protein, human; D007211:Indoles; D050838:Interferon Regulatory Factor-3; D016328:NF-kappa B; C498143:RELA protein, human; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3; D051996:Transcription Factor RelA; C081021:Go 6976; D016899:Interferon-beta; D017346:Protein Serine-Threonine Kinases; C403191:TBK1 protein, human; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D011070:Poly I-C