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1. PKC-alpha controls MYD88-dependent TLR/IL-1R signaling and cytokine production in mouse and human dendritic cells.

Author

Langlet C, Springael C, Johnson J, Thomas S, Flamand V, Leitges M, Goldman M, Aksoy E, and Willems F

Subjects
Adaptor Proteins, Vesicular Transport genetics, Adaptor Proteins, Vesicular Transport metabolism, Animals, Carbazoles pharmacology, Cell Line, Cells, Cultured, Dendritic Cells cytology, Enzyme Inhibitors pharmacology, Humans, Immunoblotting, Intracellular Signaling Peptides and Proteins metabolism, Membrane Proteins metabolism, Mice, Mice, Inbred C57BL, Mice, Inbred Strains, Mice, Knockout, Mitogen-Activated Protein Kinases metabolism, Myeloid Differentiation Factor 88 genetics, Myristoylated Alanine-Rich C Kinase Substrate, NF-kappa B metabolism, Phosphorylation, Protein Kinase C antagonists & inhibitors, Receptors, Interleukin-1 genetics, Reverse Transcriptase Polymerase Chain Reaction, Toll-Like Receptors genetics, Transcription Factor AP-1 metabolism, Cytokines metabolism, Dendritic Cells metabolism, Myeloid Differentiation Factor 88 metabolism, Protein Kinase C metabolism, Receptors, Interleukin-1 metabolism, Toll-Like Receptors metabolism
Abstract

Conventional PKC (cPKC)-alpha regulates TRIF-dependent IFN response factor 3 (IRF3)-mediated gene transcription, but its role in MyD88-dependent TLR signaling remains unknown. Herein, we demonstrate that PKC-alpha is induced by several MyD88-dependent TLR/IL-1R ligands and regulates cytokine expression in human and murine DC. First, inhibition of cPKC activity in human DC by cPKC-specific inhibitors, Gö6976 or HBDDe, downregulated the production of classical inflammatory/immunomodulatory cytokines induced by TLR2, TLR5 or IL-1R but not by TLR3 stimulation. Similarly, dominant negative PKC-alpha repressed Pam(3)CSK(4) induced NF-kappaB- and AP-1-driven promoter activities in TLR2-expressing human embryonic kidney 293 T cells. Dominant negative PKC-alpha inhibited NF-kappaB reporter activity mediated by overexpression of MyD88 but not TRIF. Unexpectedly, BM-derived DC from PKC-alpha(-/-) mice exhibited decreased TNF-alpha and IL-12p40 production induced by both MyD88- and TRIF-dependent ligands. Furthermore, PKC-alpha is coupled to TLR2 signaling proximal to MyD88 since MAPK and IkappaB kinase-alpha/beta phosphorylations and IkappaBalpha degradation were inhibited in PKC-alpha(-/-) BM-derived DC. Finally, co-immunoprecipitation assays revealed that PKC-alpha physically interacts with Pam(3)CSK(4) activated TLR2 in WT but not in MyD88(-/-) DC. Collectively this study identifies a species-specific role of PKC-alpha as a key component that controls MyD88-dependent cytokine gene expression in human and mouse but differentially regulates production of TRIF-dependent cytokines.

Published
2010
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2. Protein kinase C: a key regulator of dendritic cell function

Author

Johnson, Jolyn, Goldman, Michel, Willems, Fabienne, Bergmann, Pierre, Leo, Oberdan, Trottein, François, Bruyns, Catherine, Communi, Didier, Erneux, Christophe, and Trottein, Francois

Subjects
Interleukines, Protein kinase C, Réaction immunitaire, Interleukins, Cellules dendritiques, Cytokines, Disciplines biomédicales diverses, dendritic cells, Immune response, Protéine kinase C
Abstract

The innate immune system is an important mechanism that protects the host from infection. Viral and bacterial infection triggers activation of the transcription factors interferon response factor (IRF) 3 and nuclear factor (NF)-kB. These transcription factors collaborate to induce transcription of type I interferons (IFNs) cytokines and the interleukin (IL)-12 family of cytokines. Type I IFN and the IL-12 family of cytokines play a critical role in establishing innate immune responses as well as initiating and directing adaptive responses. Our study focused on the role of protein kinase C (PKC) isoforms in Toll-like (TLR)-dependent and –independent activation of IRF-3 and NF-kB and their subsequent regulation of IFN-beta and the IL-12 family of cytokines. TLR3, TLR4 and retinoic acid-inducible gene 1 (RIG-1)/melanoma differentiation associated gene 5 (MDA-5) activation by double stranded (ds) RNA mimic polyinosine-polycytidylic acid (poly(I:C)), lipopolysaccharide (LPS) and synthetic ds-B-DNA respectively, mediated IFN-beta as well as TNF-alpha and IL-8 synthesis in monocyte-derived DCs. Using the pharmacological inhibitor of conventional PKCs (cPKCs), Gö6976, we demonstrated that this family of kinases was involved in TLR3, TLR4 and RIG-1/ MDA-5 signaling pathways leading to the production of IFN-beta but not of TNF-alpha and IL-8. Further analysis with the use of specific kinase inactive cPKC isoforms and siRNA targeted to PKCalpha, we established that PKCalpha was the isoform involved in the TLR3 signaling pathway. In the case of TLR3, we show that PKCalphaexerts its effect downstream of TRIF and TBK1. Moreover, we show that inactivation of PKCalpha specifically inhibits the activation of IRF-3 and not that of NF-kB. Through biochemical analysis, we assessed the contribution of PKCalpha in the critical events of IRF-3 activation: a) phosphorylation b) homodimerization c) nuclear translocation d) DNA-binding and e) recruitment of creb-binding protein (CBP). We conclude that inhibition of cPKCs severely hinders the association of IRF-3 with CBP. Overall, these data revealed the critical role of cPKCs in TLR-dependent and -independent pathways leading to IFN-beta synthesis. The selective targeting of IRF-3 by cPKCs prompted us to study the possible implications of cPKCs in the transcriptional control of IL-12 family members, some of which are regulated by IRF3. Indeed, recent studies have emerged demonstrating the essential role of IRF-3 in IL-12p35 and IL-27p28 gene expression (1;2). Likewise, we investigated the role of cPKCs in the regulation of LPS- and poly(I:C)-induced expression of IL-12(p40/p35), IL-23(p40/p19) and IL-27(p28/EBI3) in monocyte-derived DCs. Treatment of monocyte-derived DCs with Gö6976 down-regulated LPS- and poly(I:C)-induced IL-12 and IL-27 synthesis while it did not alter IL-23 production. Next, we showed that impaired IL-12 and IL-27 synthesis was due to repressed IL-12p35 and IL-27p28 gene expression downstream of TLR3 and TLR4 whereas IL-23p19 and IL-27EBI3 gene expression were not modified. Reporter gene assays demonstrated that cPKCs are involved in LPS- and poly(I:C)-induced IL-12p35 and IL-27p28 promoter activity. Finally, experiments in bone marrow-derived DCs from IRF-3-/- and wild type mice showed that IL-23 synthesis does not require IRF-3 activation. We conclude that cPKCs through the control of IRF-3 activity are critically involved in the regulation of IL-12 and IL-27 synthesis downstream of TLR3 and TLR4 while they do not participate in IRF-3-independent IL-23 synthesis. On whole, we demonstrated a novel function for cPKCs in the regulation of IRF-3 and IRF-3 dependent gene expression, specifically IFN-beta, IL-12 and IL-27. In light of the important and divergent roles of IFN-beta and IL-12 family of cytokines on the development of T helper (Th) Th1, Th2, Th17-mediated immune responses, cPKCs represent a potential target for therapeutic immunomodulation. This modulation needs to be carefully administered due to the complex interplay of the IL-12 family members in immunity., Doctorat en sciences biomédicales, info:eu-repo/semantics/published

Published
2007

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