Kristi L. Peters

Novel roles of TLR3 tyrosine phosphorylation and PI3 kinase in double-stranded RNA signaling.

Double-stranded RNA (dsRNA), a frequent byproduct of virus infection, is recognized by Toll-like receptor 3 (TLR3) to mediate innate immune response to virus infection. TLR3 signaling activates the transcription factor IRF-3 by its Ser/Thr phosphorylation, accompanied by its dimerization and nuclear translocation. It has been reported that the Ser/Thr kinase TBK-1 is essential for TLR3-mediated activation and phosphorylation of IRF-3. Here we report that dsRNA-activated phosphorylation of two specific tyrosine residues of TLR3 is essential for initiating two distinct signaling pathways. One involves activation of TBK-1 and the other recruits and activates PI3 kinase and the downstream kinase, Akt, leading to full phosphorylation and activation of IRF-3. When PI3 kinase is not recruited to TLR3 or its activity is blocked, IRF-3 is only partially phosphorylated and fails to bind the promoter of the target gene in dsRNA-treated cells. Thus, the PI3K-Akt pathway plays an essential role in TLR3-mediated gene induction.

A critical role for IRAK4 kinase activity in Toll-like receptor–mediated innate immunity

IRAK4 is a member of IL-1 receptor (IL-1R)–associated kinase (IRAK) family and has been shown to play an essential role in Toll-like receptor (TLR)–mediated signaling. We recently generated IRAK4 kinase-inactive knock-in mice to examine the role of kinase activity of IRAK4 in TLR-mediated signaling pathways. The IRAK4 kinase–inactive knock-in mice were completely resistant to lipopolysaccharide (LPS)- and CpG-induced shock, due to impaired TLR-mediated induction of proinflammatory cytokines and chemokines. Although inactivation of IRAK4 kinase activity did not affect the levels of TLR/IL-1R–mediated nuclear factor κB activation, a reduction of LPS-, R848-, and IL-1–mediated mRNA stability contributed to the reduced cytokine and chemokine production in bone marrow–derived macrophages from IRAK4 kinase–inactive knock-in mice. Both TLR7- and TLR9-mediated type I interferon production was abolished in plasmacytoid dendritic cells isolated from IRAK4 knock-in mice. In addition, influenza virus–induced production of interferons in plasmacytoid DCs was also dependent on IRAK4 kinase activity. Collectively, our results indicate that IRAK4 kinase activity plays a critical role in TLR-dependent immune responses.

Viral apoptosis is induced by IRF-3-mediated activation of Bax

Upon infection with many RNA viruses, the cytoplasmic retinoic acid inducible gene‐I (RIG‐I) pathway activates the latent transcription factor IRF‐3, causing its nuclear translocation and the induction of many antiviral genes, including those encoding interferons. Here, we report a novel and distinct activity of IRF‐3, in virus‐infected cells, that induces apoptosis. Using genetically defective mouse and human cell lines, we demonstrated that, although both pathways required the presence of RIG‐I, IPS1, TRAF3 and TBK1, only the apoptotic pathway required the presence of TRAF2 and TRAF6 in addition. More importantly, transcriptionally inactive IRF‐3 mutants, such as the one missing its DNA‐binding domain, could efficiently mediate apoptosis. Apoptosis was triggered by the direct interaction of IRF‐3, through a newly identified BH3 domain, with the pro‐apoptotic protein Bax, their co‐translocation to the mitochondria and the resulting activation of the mitochondrial apoptotic pathway. Thus, IRF‐3 is a dual‐action cytoplasmic protein that, upon activation, translocates to the nucleus or to the mitochondrion and triggers two complementary antiviral responses of the infected cell.

IRF-3-dependent, NFκB- and JNK-independent activation of the 561 and IFN-β genes in response to double-stranded RNA

Double-stranded (ds) RNA induces transcription of the 561 gene by activating IFN regulatory factor (IRF) transcription factors, whereas similar induction of the IFN-β gene is thought to require additional activation of NFκB and AP-1. In mutant P2.1 cells, dsRNA failed to activate NFκB, IRF-3, p38, or c-Jun N-terminal kinase, and transcription of neither 561 mRNA nor IFN-β mRNA was induced. The defect in the IRF-3 pathway was traced to a low cellular level of this protein because of its higher rate of degradation in P2.1 cells. As anticipated, in several clonal derivatives of P2.1 cells expressing different levels of transfected IRF-3, activation of IRF-3 and induction of 561 mRNA by dsRNA was restored fully, although the defects in other responses to dsRNA persisted. Surprisingly, IFN-β mRNA also was induced strongly in these cells in response to dsRNA, demonstrating that the activation of NFκB and AP-1 is not required. This conclusion was confirmed in wild-type cells overexpressing IRF-3 by blocking NFκB activation with the IκB superrepressor and AP-1 activation with a p38 inhibitor. Therefore, IRF-3 activation by dsRNA is sufficient to induce the transcription of genes with simple promoters such as 561 as well as complex promoters such as IFN-β.

Two Tyrosine Residues of Toll-like Receptor 3 Trigger Different Steps of NF-κB Activation

Innate immune response to viral infection is often triggered by Toll-like receptor 3 (TLR3)-mediated signaling by double-stranded (ds) RNA, which culminates in the activation of the transcription factor NF-κB and induction of NF-κB-driven genes. We demonstrated that dsRNA-induced phosphorylation of two specific tyrosine residues, 759 and 858, of TLR3 was necessary and sufficient for complete activation of the NF-κB pathway. When Tyr-759 of TLR3 was mutated, gene induction was inhibited, although NF-κB was partially activated. It was released from IκB and translocated to the nucleus but failed to bind to the κB site of the target A20 gene promoter. This defect could be attributed to incomplete phosphorylation of the RelA (p65) subunit of NF-κB, as revealed by two-dimensional gel analyses of p65, isolated from dsRNA-treated cells expressing either wild type TLR3 or the Tyr-759 → Phe mutant TLR3. Thus, two phosphotyrosine residues of TLR3 activate two distinct pathways, one leading to NF-κB release and the other leading to its phosphorylation.

Abstract C153: Both TRAIL receptor DR5 isoforms are required for the induction of cell death in human colon carcinoma

Colorectal cancer continues to be the third most common malignancy in the United States. This disease remains resistant to treatment, with only 25–30% of patients disease‐free long term; for the rest, median su rvival is only 18–22 months. We previously demonstrated synergistic interaction between FUra/LV combined with IFN‐ (which potentiates FUra/LV‐induced DNA damage and requires the death receptor Fas) in human colon carcinoma cell lines and xenografts. FUra/LV/IFN‐ also demonstrated responses in heavily pretreated and previously untreated patients in Phase I trial and is currently completing Phase II evaluation in Stage IV colorectal cancer. Further, lexatumumab, an agonistic TRAIL‐R2 (DR5) monoclonal antibody, is highly synergistic with FUra/LV/IFN‐ in xenograft models, dependent on TRAIL receptor activation. Lexatumumab demonstrated the broadest spectrum in vitro activity in a panel of 6 human colon carcinoma cell lines when compared to the activity of the recombinant ligand, TRAIL (targeting DR4 and DR5). To further understand the mechanism of lexatumumab‐induced cell death via DR5, DR5 expression, was determined in human colon carcinoma cell lines (Western, RT‐PCR) and primary colon tumors (RT‐PCR). The short isoform of the receptor (DR5S) was expressed at a higher level than the long isoform (DR5L). To determine the specific function of each isoform in regulating death signaling, a BJAB‐derived cell line lacking DR5 expression (DR5−/−) was employed. DR5 −/− cells were resistant to lexatumumab‐induced apoptosis, even when each DR5 isoform was expressed individually, despite the fact that the individual isoforms could still bind ligand (lexatumumab). Analysis of receptor complex formation indicated that DR5, FADD, and caspase‐8 were recruited; however, caspase‐8 was not processed. Apoptotic signaling was restored when both DR5 isoforms were re‐expressed in DR5−/− cells. Lexatumumab sensitivity of HCT8 cells was eliminated by stable shRNA knockdown of both DR5 isoforms, as was the recruitment of FADD and caspase‐8 to the receptor complex. In contrast, following 1 hr lexatumumab treatment in wild type HCT8 cells, both DR5 isoforms were recruited to the receptor complex; cell death correlated with recruitment of FADD, cleavage of caspase‐8, cleavage of RIP, and reduced expression of c‐FLIPS and c‐FLIPL. TRAF2 and c‐IAP2, but not TRAF1 or c‐IAP1, were also recruited; however, the significance of these signaling molecules in the cell death response is currently unknown. Overall, data indicate that both DR5S and DR5L are required for lexatumumab‐induced cell death via the DR5 receptor. Although the significance of this is currently unknown, factors that regulate lexatumumab‐induced cell death vs survival at the level of receptor complexes are currently being elucidated. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C153.

IRF-3 activation by Sendai virus infection is required for cellular apoptosis and avoidance of persistence (Journal of Virology (2008) 82, 7, (3500-3508))

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