Adrian T. Ting

The tumour suppressor cyld is a negative regulator of rig-i-mediated antiviral response

On detecting viral RNAs, the RNA helicase retinoic acid‐inducible gene I (RIG‐I) activates the interferon regulatory factor 3 (IRF3) signalling pathway to induce type I interferon (IFN) gene transcription. How this antiviral signalling pathway might be negatively regulated is poorly understood. Microarray and bioinformatic analysis indicated that the expression of RIG‐I and that of the tumour suppressor CYLD (cylindromatosis), a deubiquitinating enzyme that removes Lys 63‐linked polyubiquitin chains, are closely correlated, suggesting a functional association between the two molecules. Ectopic expression of CYLD inhibits the IRF3 signalling pathway and IFN production triggered by RIG‐I; conversely, CYLD knockdown enhances the response. CYLD removes polyubiquitin chains from RIG‐I as well as from TANK binding kinase 1 (TBK1), the kinase that phosphorylates IRF3, coincident with an inhibition of the IRF3 signalling pathway. Furthermore, CYLD protein level is reduced in the presence of tumour necrosis factor and viral infection, concomitant with enhanced IFN production. These findings show that CYLD is a negative regulator of RIG‐I‐mediated innate antiviral response.

Ubiquitination of RIP1 regulates an NF-κB-independent cell death switch in TNF signaling

TNF receptor 1 (TNFR1) can trigger opposing responses within the same cell: a prosurvival response or a cell-death pathway [1, 2]. Cell survival requires NF-kappaB-mediated transcription of prosurvival genes [3-9]; apoptosis occurs if NF-kappaB signaling is blocked [5, 7-9]. Hence, activation of NF-kappaB acts as a cell-death switch during TNF signaling. This study demonstrates that the pathway includes another cell-death switch that is independent of NF-kappaB. We show that lysine 63-linked ubiquitination of RIP1 on lysine 377 inhibits TNF-induced apoptosis first through an NF-kappaB-independent mechanism and, subsequently, through an NF-kappaB-dependent mechanism. In contrast, in the absence of ubiquitination, RIP1 serves as a proapoptotic signaling molecule by engaging CASPASE-8. Therefore, RIP1 is a dual-function molecule that can be either prosurvival or prodeath depending on its ubiquitination state, and this serves as an NF-kappaB-independent cell-death switch early in TNF signaling. These results provide an explanation for the conflicting reports on the role of RIP1 in cell death; this role was previously suggested to be both prosurvival and prodeath [10-12]. Because TRAF2 is the E3 ligase for RIP1 [13], these observations provide an explanation for the NF-kappaB-independent antiapoptotic function previously described for TRAF2 [14-16].

A20 Inhibits Tumor Necrosis Factor (TNF) Alpha-Induced Apoptosis by Disrupting Recruitment of TRADD and RIP to the TNF Receptor 1 Complex in Jurkat T Cells

ABSTRACT Tumor necrosis factor receptor 1 (TNFR1) can trigger distinct signaling pathways leading to either the activation of NF-κB transcription factors or apoptosis. NF-κB activation results in the expression of antiapoptotic genes that inhibit the apoptosis pathway that is activated in parallel. However, the molecular mechanism of this inhibition remains poorly characterized. We have isolated a Jurkat T-cell mutant that exhibits enhanced sensitivity to TNF-induced apoptosis as a result of a deficiency in I-κB kinase γ (IKKγ)/NEMO, an essential component of the IKK complex and NF-κB pathway. We show here that the zinc finger protein A20 is an NF-κB-inducible gene that can protect the IKKγ-deficient cells from TNF-induced apoptosis by disrupting the recruitment of the death domain signaling molecules TRADD and RIP to the receptor signaling complex. Our study, together with reports on the role of other antiapoptotic proteins such as c-FLIP and c-IAP, suggests that, in order to ensure an effective shutdown of the apoptotic pathway, TNF induces multiple NF-κB-dependent genes that inhibit successive steps in the TNFR1 death signaling pathway.

Inhibition of MAPK and NF-κB Pathways Is Necessary for Rapid Apoptosis in Macrophages Infected with Yersinia

Macrophages respond to infection with pathogenic Yersinia species by activating MAPK- and NF-κB-signaling pathways. To counteract this response, Yersiniae secrete a protease (Yersinia outer protein J (YopJ)) that is delivered into macrophages, deactivates MAPK- and NF-κB-signaling pathways, and induces apoptosis. NF-κB promotes cell survival by up-regulating expression of several apoptosis inhibitor genes. Previous studies show that deactivation of the NF-κB pathway by YopJ is important for Yersinia-induced apoptosis. To determine whether deactivation of the NF-κB pathway is sufficient for Yersinia-induced apoptosis, two inhibitors of the NF-κB pathway, IκBα superrepressor or A20, were expressed in macrophages. Macrophages expressing these proteins were infected with Yersinia pseudotuberculosis strains that secrete functionally active or inactive forms of YopJ. Apoptosis levels were substantially higher (5- to 10-fold) when active YopJ was delivered into macrophages expressing IκBα superrepressor or A20, suggesting that deactivation of the NF-κB pathway is not sufficient for rapid Yersinia-induced apoptosis. When macrophages expressing A20 were treated with specific inhibitors of MAPKs, similar levels of apoptosis (within ∼2-fold) were observed when active or inactive YopJ were delivered during infection. These results suggest that MAPK and NF-κB pathways function together to up-regulate apoptosis inhibitor gene expression in macrophages in response to Yersinia infection and that YopJ deactivates both pathways to promote rapid apoptosis. In addition, treating macrophages with a proteasome inhibitor results in higher levels of infection-induced apoptosis than can be achieved by blocking NF-κB function alone, suggesting that proapoptotic proteins are stabilized when proteasome function is blocked in macrophages.

B Cell Maturation Antigen, the Receptor for a Proliferation-Inducing Ligand and B Cell-Activating Factor of the TNF Family, Induces Antigen Presentation in B Cells

B cell maturation Ag (BCMA), a member of the TNFR superfamily expressed on B cells, binds to a proliferation-inducing ligand (APRIL) and B cell-activating factor of the TNF family (BAFF) but the specific B cell responses regulated by BCMA remain unclear. This study demonstrates that ligation of A20 B cells transfected with BCMA induces the expression of CD40, CD80/B7-1, CD86/B7-2, MHC class II, and CD54/ICAM-1, which subsequently enhances the presentation of OVA peptide Ag to DO11.10 T cells. BCMA expression in murine splenic B cells can be induced with IL-4 and IL-6, allowing subsequent treatment with APRIL or agonist anti-BCMA to similarly induce Ag presentation. A comparative analysis of hybrid receptors of TNFR2 fused to the cytoplasmic domains of APRIL/BAFF receptors found that only BCMA, but not transmembrane activator and calcium-modulator and cyclophilin ligand interactor or BAFF-R, is capable of activating Ag presentation. Although all three receptors can trigger NF-κB signaling, only BCMA activates the JNK pathway conferring on BCMA the specific ability to activate this Ag presentation response.

Induction of Diabetes in Aged C57B6 Mice Results in Severe Nephropathy : An Association with Oxidative Stress, Endoplasmic Reticulum Stress, and Inflammation

Kidney aging is a slowly progressive process that is postulated to be accelerated by intervening diseases, such as diabetes, due in part to the addition of excessive stress and inflammation from the intervening disease to the underlying aging process. This hypothesis was tested by inducing diabetes with streptozotocin in 18-month-old, aging mice. After 4 months of diabetes, these mice developed severe albuminuria, elevated creatinine levels, and renal lesions including extensive apoptotic cell death, glomerulosclerosis, afferent and efferent hyalinosis, and tubulointerstitial inflammation and fibrosis. These symptoms were associated with elevated oxidative stress. The presence of endoplasmic reticulum (ER) stress in 22-month-old diabetic kidneys resulted in up-regulation of C/EBP homologous protein (CHOP), which may play a role in increasing kidney lesions because CHOP-deficient proximal tubular cells were resistant to ER stress-induced cell death, and CHOP-deficient mice were protected from diabetic nephropathy. Moreover, CHOP-deficient mice did not develop albuminuria as they aged. Inflammation, another key component of progressive diabetic nephropathy, was prominent in 22-month-old diabetic kidneys. The expression of tumor-necrosis factor-alpha in 22-month-old diabetic kidneys may play a role in inflammation, ER stress, and apoptosis. Thus, diabetes may accelerate the underlying kidney aging process present in old mice.

Epstein-Barr virus-induced gene 3 negatively regulates IL-17, IL-22 and RORγt

Epstein‐Barr virus‐induced gene 3 (EBI3) associates with p28 to form IL‐27 and with IL‐12p35 to form IL‐35. IL‐27Rα–/– mice studies indicate that IL‐27 negatively regulates Th17 cell differentiation. However, no EBI3, p28 or p35‐deficiency studies that directly address the role of EBI3, p28 or p35 on Th17 cells have been done. Here, we demonstrate that spleen cells derived from EBI3–/– mice produce significantly higher levels of IL‐17 as well as IL‐22 upon stimulation with OVA. In vitro derived EBI3–/– Th17 cells also produced significantly higher levels of IL‐17 and IL‐22 than WT cells. The frequency of IL‐17‐producing cells was also elevated when EBI3–/– cells were cultured under Th17 conditions. In addition, spleen cells from EBI3–/– mice immunized with Listeria monocytogenes produced significantly elevated levels of IL‐17 and IL‐22. Furthermore, the Th17 transcription factor RORγt was significantly enhanced in EBI3–/– cells. Finally, EBI3–/– mice exhibited a reduced bacterial load following an acute challenge with L. monocytogenes or a re‐challenge of previously immunized mice, suggesting that EBI3 negatively regulates both innate and adaptive immunity. Taken together, these data provide direct evidence that EBI3 negatively regulates the expression of IL‐17, IL‐22 and RORγt as well as protective immunity against L. monocytogenes.

NEMO/IKKγ regulates an early NF-κB-independent cell death checkpoint during TNF signaling

TNF receptor 1 (TNFR1) ligation can result in cell survival or cell death. What determines which of the two opposing responses is triggered is not fully understood. The current model suggests that it is the activation of the NF-κB pathway and its induction of prosurvival genes, or the lack thereof, which determines the outcome. NF-κB essential modifier (NEMO)/IκB kinase-γ (IKKγ)-deficient cells are highly sensitive to apoptosis, and as NEMO is essential for NF-κB activation, it has been assumed that this is due to the lack of NF-κB. This study demonstrates that this assumption was incorrect and that NEMO has another antiapoptotic function that is independent of its role in the NF-κB pathway. NEMO prevents receptor interacting protein-1 (RIP1) from engaging CASPASE-8 before NF-κB-mediated induction of antiapoptotic genes. Without NEMO, RIP1 associates with CASPASE-8 resulting in rapid tumor necrosis factor (TNF)-induced apoptosis. These results suggest that there are two cell-death checkpoints following TNF stimulation: an early transcription-independent checkpoint whereby NEMO restrains RIP1 from activating the caspase cascade, followed by a later checkpoint dependent on NF-κB-mediated transcription of prosurvival genes.

Therapeutic potential and molecular mechanism of a novel, potent, nonpeptide, Smac mimetic SM-164 in combination with TRAIL for cancer treatment

Smac mimetics are being developed as a new class of anticancer therapies. Because the single-agent activity of Smac mimetics is very limited, rational combinations represent a viable strategy for their clinical development. The combination of Smac mimetics with TNF-related apoptosis inducing ligand (TRAIL) may be particularly attractive because of the low toxicity of TRAIL to normal cells and the synergistic antitumor activity observed for the combination. In this study, we have investigated the combination synergy between TRAIL and a potent Smac mimetic, SM-164, in vitro and in vivo and the underlying molecular mechanism of action for the synergy. Our study shows that SM-164 is highly synergistic with TRAIL in vitro in both TRAIL-sensitive and TRAIL-resistant cancer cell lines of breast, prostate, and colon cancer. Furthermore, the combination of SM-164 with TRAIL induces rapid tumor regression in vivo in a breast cancer xenograft model in which either agent is ineffective. Our data show that X-linked IAP (XIAP) and cellular IAP 1 (cIAP1), but not cIAP2, work in concert to attenuate the activity of TRAIL; SM-164 strongly enhances TRAIL activity by concurrently targeting XIAP and cIAP1. Moreover, although RIP1 plays a minimal role in the activity of TRAIL as a single agent, it is required for the synergistic interaction between TRAIL and SM-164. This study provides a strong rationale to develop the combination of SM-164 and TRAIL as a new therapeutic strategy for the treatment of human cancer. Mol Cancer Ther; 10(5); 902–14. ©2011 AACR.

NEMO inhibits programmed necrosis in an NFκB-independent manner by restraining RIP1.

TNF can trigger two opposing responses: cell survival and cell death. TNFR1 activates caspases that orchestrate apoptosis but some cell types switch to a necrotic death when treated with caspase inhibitors. Several genes that are required to orchestrate cell death by programmed necrosis have been identified, such as the kinase RIP1, but very little is known about the inhibitory signals that keep this necrotic cell death pathway in check. We demonstrate that T cells lacking the regulatory subunit of IKK, NFκB essential modifier (NEMO), are hypersensitive to programmed necrosis when stimulated with TNF in the presence of caspase inhibitors. Surprisingly, this pro-survival activity of NEMO is independent of NFκB-mediated gene transcription. Instead, NEMO inhibits necrosis by binding to ubiquitinated RIP1 to restrain RIP1 from engaging the necrotic death pathway. In the absence of NEMO, or if ubiquitination of RIP1 is blocked, necrosis ensues when caspases are blocked. These results indicate that recruitment of NEMO to ubiquitinated RIP1 is a key step in the TNFR1 signaling pathway that determines whether RIP1 triggers a necrotic death response.