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Regulation of Toll-like receptor signaling by NDP52-mediated selective autophagy is normally inactivated by A20.

Inomata M, Niida S, Shibata K, Into T - Cell. Mol. Life Sci. (2011)

Bottom Line: However, this autophagy was not associated with canonical autophagic processes, including involvement of Beclin-1 and conversion of LC3-I to LC3-II.NDP52 was polyubiquitinated by TRAF6 and was involved in aggregation of TRAF6, which may result in the selective degradation.Furthermore, although A20 is known as a signaling fine-tuner to prevent excess TLR signaling, it paradoxically downregulates the fine-tuning effect of NDP52 on TLR signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Microbiology, Division of Oral Infections and Health Sciences, Asahi University School of Dentistry, Hozumi 1851, Mizuho, Gifu 501-0296, Japan.

ABSTRACT
Toll-like receptor (TLR) signaling is linked to autophagy that facilitates elimination of intracellular pathogens. However, it is largely unknown whether autophagy controls TLR signaling. Here, we report that poly(I:C) stimulation induces selective autophagic degradation of the TLR adaptor molecule TRIF and the signaling molecule TRAF6, which is revealed by gene silencing of the ubiquitin-editing enzyme A20. This type of autophagy induced formation of autophagosomes and could be suppressed by an autophagy inhibitor and lysosomal inhibitors. However, this autophagy was not associated with canonical autophagic processes, including involvement of Beclin-1 and conversion of LC3-I to LC3-II. Through screening of TRIF-interacting 'autophagy receptors' in human cells, we identified that NDP52 mediated the selective autophagic degradation of TRIF and TRAF6 but not TRAF3. NDP52 was polyubiquitinated by TRAF6 and was involved in aggregation of TRAF6, which may result in the selective degradation. Intriguingly, only under the condition of A20 silencing, NDP52 could effectively suppress poly(I:C)-induced proinflammatory gene expression. Thus, this study clarifies a selective autophagic mechanism mediated by NDP52 that works downstream of TRIF-TRAF6. Furthermore, although A20 is known as a signaling fine-tuner to prevent excess TLR signaling, it paradoxically downregulates the fine-tuning effect of NDP52 on TLR signaling.

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NDP52 negatively regulates TRIF-triggered transcriptional activities of NF-κB and IRF3. a, b HEK293T cells were transfected with Flag-TRIF and pNF-κB-Luc (a) or p561-luc (b) together with indicated amounts of HA-NDP52 for 24 h. Then, luciferase activity was measured. Data are expressed as the mean ± SD (n = 3). c, d HEK293-TLR4/MD2-CD14 cells were transfected with pNF-κB-Luc (c) or p561-luc (d) together with indicated amounts of HA-NDP52 for 18 h. Cells were stimulated with 100 ng/ml E. coli LPS for 6 h. Then, luciferase activity was measured. Data are expressed as the mean ± SD (n = 3). e HEK293T cells were transfected with Flag-TRIF and pNF-κB-Luc together with HA-NDP52 or empty vector (Mock) for 18 h. Cells were treated with 10 mM 3-MA for 6 h. Then, luciferase activity was measured. Data are expressed as the mean ± SD (n = 3). *P < 0.01, for comparison with the value of DMSO. f HEK293T cells were transfected with control siRNA or Beclin-1 siRNA for 24 h. Cells were further transfected with Flag-TRIF, pNF-κB-Luc together with HA-NDP52 or empty vector (Mock) for 18 h. Then, luciferase activity was measured. Data are expressed as the mean ± SD (n = 3). All results are representative of three independent experiments
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Fig5: NDP52 negatively regulates TRIF-triggered transcriptional activities of NF-κB and IRF3. a, b HEK293T cells were transfected with Flag-TRIF and pNF-κB-Luc (a) or p561-luc (b) together with indicated amounts of HA-NDP52 for 24 h. Then, luciferase activity was measured. Data are expressed as the mean ± SD (n = 3). c, d HEK293-TLR4/MD2-CD14 cells were transfected with pNF-κB-Luc (c) or p561-luc (d) together with indicated amounts of HA-NDP52 for 18 h. Cells were stimulated with 100 ng/ml E. coli LPS for 6 h. Then, luciferase activity was measured. Data are expressed as the mean ± SD (n = 3). e HEK293T cells were transfected with Flag-TRIF and pNF-κB-Luc together with HA-NDP52 or empty vector (Mock) for 18 h. Cells were treated with 10 mM 3-MA for 6 h. Then, luciferase activity was measured. Data are expressed as the mean ± SD (n = 3). *P < 0.01, for comparison with the value of DMSO. f HEK293T cells were transfected with control siRNA or Beclin-1 siRNA for 24 h. Cells were further transfected with Flag-TRIF, pNF-κB-Luc together with HA-NDP52 or empty vector (Mock) for 18 h. Then, luciferase activity was measured. Data are expressed as the mean ± SD (n = 3). All results are representative of three independent experiments

Mentions: Degradation of signaling molecules is generally thought to be linked to downregulation of signal transduction. We therefore investigated whether NDP52 is able to negatively regulate TRIF-mediated signaling. In HEK293T cells, transfection of NDP52 could attenuate the TRIF overexpression-induced transcriptional activities of NF-κB and IRF3 (Fig. 5a, b). In addition, in HEK293 cells stably expressing TLR4/MD-2, transfection of NDP52 attenuated the LPS-induced activation of NF-κB and IRF3 (Fig. 5c, d). Moreover, treatment of cells with 3-MA potentiated TRIF-induced activation of NF-κB, and attenuated the negative regulatory effect of NDP52 (Fig. 5e). In addition, consistent with the result shown in Fig. 2h, silencing of Beclin-1 did not affect the negative regulatory effect of NDP52 (Fig. 5f). Thus, NDP52-mediated autophagic degradation has a potential to negatively regulate TRIF-mediated signaling.Fig. 5


Regulation of Toll-like receptor signaling by NDP52-mediated selective autophagy is normally inactivated by A20.

Inomata M, Niida S, Shibata K, Into T - Cell. Mol. Life Sci. (2011)

NDP52 negatively regulates TRIF-triggered transcriptional activities of NF-κB and IRF3. a, b HEK293T cells were transfected with Flag-TRIF and pNF-κB-Luc (a) or p561-luc (b) together with indicated amounts of HA-NDP52 for 24 h. Then, luciferase activity was measured. Data are expressed as the mean ± SD (n = 3). c, d HEK293-TLR4/MD2-CD14 cells were transfected with pNF-κB-Luc (c) or p561-luc (d) together with indicated amounts of HA-NDP52 for 18 h. Cells were stimulated with 100 ng/ml E. coli LPS for 6 h. Then, luciferase activity was measured. Data are expressed as the mean ± SD (n = 3). e HEK293T cells were transfected with Flag-TRIF and pNF-κB-Luc together with HA-NDP52 or empty vector (Mock) for 18 h. Cells were treated with 10 mM 3-MA for 6 h. Then, luciferase activity was measured. Data are expressed as the mean ± SD (n = 3). *P < 0.01, for comparison with the value of DMSO. f HEK293T cells were transfected with control siRNA or Beclin-1 siRNA for 24 h. Cells were further transfected with Flag-TRIF, pNF-κB-Luc together with HA-NDP52 or empty vector (Mock) for 18 h. Then, luciferase activity was measured. Data are expressed as the mean ± SD (n = 3). All results are representative of three independent experiments
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Fig5: NDP52 negatively regulates TRIF-triggered transcriptional activities of NF-κB and IRF3. a, b HEK293T cells were transfected with Flag-TRIF and pNF-κB-Luc (a) or p561-luc (b) together with indicated amounts of HA-NDP52 for 24 h. Then, luciferase activity was measured. Data are expressed as the mean ± SD (n = 3). c, d HEK293-TLR4/MD2-CD14 cells were transfected with pNF-κB-Luc (c) or p561-luc (d) together with indicated amounts of HA-NDP52 for 18 h. Cells were stimulated with 100 ng/ml E. coli LPS for 6 h. Then, luciferase activity was measured. Data are expressed as the mean ± SD (n = 3). e HEK293T cells were transfected with Flag-TRIF and pNF-κB-Luc together with HA-NDP52 or empty vector (Mock) for 18 h. Cells were treated with 10 mM 3-MA for 6 h. Then, luciferase activity was measured. Data are expressed as the mean ± SD (n = 3). *P < 0.01, for comparison with the value of DMSO. f HEK293T cells were transfected with control siRNA or Beclin-1 siRNA for 24 h. Cells were further transfected with Flag-TRIF, pNF-κB-Luc together with HA-NDP52 or empty vector (Mock) for 18 h. Then, luciferase activity was measured. Data are expressed as the mean ± SD (n = 3). All results are representative of three independent experiments
Mentions: Degradation of signaling molecules is generally thought to be linked to downregulation of signal transduction. We therefore investigated whether NDP52 is able to negatively regulate TRIF-mediated signaling. In HEK293T cells, transfection of NDP52 could attenuate the TRIF overexpression-induced transcriptional activities of NF-κB and IRF3 (Fig. 5a, b). In addition, in HEK293 cells stably expressing TLR4/MD-2, transfection of NDP52 attenuated the LPS-induced activation of NF-κB and IRF3 (Fig. 5c, d). Moreover, treatment of cells with 3-MA potentiated TRIF-induced activation of NF-κB, and attenuated the negative regulatory effect of NDP52 (Fig. 5e). In addition, consistent with the result shown in Fig. 2h, silencing of Beclin-1 did not affect the negative regulatory effect of NDP52 (Fig. 5f). Thus, NDP52-mediated autophagic degradation has a potential to negatively regulate TRIF-mediated signaling.Fig. 5

Bottom Line: However, this autophagy was not associated with canonical autophagic processes, including involvement of Beclin-1 and conversion of LC3-I to LC3-II.NDP52 was polyubiquitinated by TRAF6 and was involved in aggregation of TRAF6, which may result in the selective degradation.Furthermore, although A20 is known as a signaling fine-tuner to prevent excess TLR signaling, it paradoxically downregulates the fine-tuning effect of NDP52 on TLR signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Microbiology, Division of Oral Infections and Health Sciences, Asahi University School of Dentistry, Hozumi 1851, Mizuho, Gifu 501-0296, Japan.

ABSTRACT
Toll-like receptor (TLR) signaling is linked to autophagy that facilitates elimination of intracellular pathogens. However, it is largely unknown whether autophagy controls TLR signaling. Here, we report that poly(I:C) stimulation induces selective autophagic degradation of the TLR adaptor molecule TRIF and the signaling molecule TRAF6, which is revealed by gene silencing of the ubiquitin-editing enzyme A20. This type of autophagy induced formation of autophagosomes and could be suppressed by an autophagy inhibitor and lysosomal inhibitors. However, this autophagy was not associated with canonical autophagic processes, including involvement of Beclin-1 and conversion of LC3-I to LC3-II. Through screening of TRIF-interacting 'autophagy receptors' in human cells, we identified that NDP52 mediated the selective autophagic degradation of TRIF and TRAF6 but not TRAF3. NDP52 was polyubiquitinated by TRAF6 and was involved in aggregation of TRAF6, which may result in the selective degradation. Intriguingly, only under the condition of A20 silencing, NDP52 could effectively suppress poly(I:C)-induced proinflammatory gene expression. Thus, this study clarifies a selective autophagic mechanism mediated by NDP52 that works downstream of TRIF-TRAF6. Furthermore, although A20 is known as a signaling fine-tuner to prevent excess TLR signaling, it paradoxically downregulates the fine-tuning effect of NDP52 on TLR signaling.

Show MeSH
Related in: MedlinePlus