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TRAM is involved in IL-18 signaling and functions as a sorting adaptor for MyD88.

Ohnishi H, Tochio H, Kato Z, Kawamoto N, Kimura T, Kubota K, Yamamoto T, Funasaka T, Nakano H, Wong RW, Shirakawa M, Kondo N - PLoS ONE (2012)

Bottom Line: These findings suggest that TRAM serves as the sorting adaptor for MyD88 in IL-18 signaling, which then facilitates the signal transduction.The binding sites for TRAM are located in the TIR domain of MyD88 and actually overlap with the binding sites for Mal.MyD88, the multifunctional signaling adaptor that works together with most of the TLR members and with the IL-1/IL-18 receptors, can interact with two distinct sorting adaptors, TRAM and Mal, in a conserved manner in a distinct context.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Japan. ohnishih@gifu-u.ac.jp

ABSTRACT
MyD88, a Toll/interleukin-1 receptor homology (TIR) domain-containing adaptor protein, mediates signals from the Toll-like receptors (TLR) or IL-1/IL-18 receptors to downstream kinases. In MyD88-dependent TLR4 signaling, the function of MyD88 is enhanced by another TIR domain-containing adaptor, Mal/TIRAP, which brings MyD88 to the plasma membrane and promotes its interaction with the cytosolic region of TLR4. Hence, Mal is recognized as the "sorting adaptor" for MyD88. In this study, a direct interaction between MyD88-TIR and another membrane-sorting adaptor, TRAM/TICAM-2, was demonstrated in vitro. Cell-based assays including RNA interference experiments and TRAM deficient mice revealed that the interplay between MyD88 and TRAM in cells is important in mediating IL-18 signal transduction. Live cell imaging further demonstrated the co-localized accumulation of MyD88 and TRAM in the membrane regions in HEK293 cells. These findings suggest that TRAM serves as the sorting adaptor for MyD88 in IL-18 signaling, which then facilitates the signal transduction. The binding sites for TRAM are located in the TIR domain of MyD88 and actually overlap with the binding sites for Mal. MyD88, the multifunctional signaling adaptor that works together with most of the TLR members and with the IL-1/IL-18 receptors, can interact with two distinct sorting adaptors, TRAM and Mal, in a conserved manner in a distinct context.

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The knock-down effects of TRAM in IL-18 or LPS/TLR4 signaling.(A) RT-PCR of the TRAM mRNA knock-down in HEK293T cells by the psiRNA-TICAM-2 but not by the nonspecific scrambled sequence coded psiRNA vector. “-“ indicates cells not transfected with siRNA; “Actb” indicates the loading control (β-actin mRNA). (B–E) The effect of the knock-down of TRAM using shRNA or the dominant negative form of TRAM (C117H) for the IL-18 or LPS induced NF-κB or IFN-β-promoter luciferase activity assay. The NF-κB and IFN-β-promoter activities with IL-18 or LPS stimulation were significantly decreased in the IL-18Rβ co-transfected HEK293T cells or HEK293-hTLR4-MD2-CD14 cells.
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pone-0038423-g003: The knock-down effects of TRAM in IL-18 or LPS/TLR4 signaling.(A) RT-PCR of the TRAM mRNA knock-down in HEK293T cells by the psiRNA-TICAM-2 but not by the nonspecific scrambled sequence coded psiRNA vector. “-“ indicates cells not transfected with siRNA; “Actb” indicates the loading control (β-actin mRNA). (B–E) The effect of the knock-down of TRAM using shRNA or the dominant negative form of TRAM (C117H) for the IL-18 or LPS induced NF-κB or IFN-β-promoter luciferase activity assay. The NF-κB and IFN-β-promoter activities with IL-18 or LPS stimulation were significantly decreased in the IL-18Rβ co-transfected HEK293T cells or HEK293-hTLR4-MD2-CD14 cells.

Mentions: After obtaining the evidence that TRAM interacts with MyD88 both in vitro and in cells, we then examined the possible involvement of TRAM in IL-18 signaling. We knocked-down the endogenous TRAM expression in HEK293 cells using siRNA techniques and then performed NF-κB reporter assays for the IL-18 signal transduction in the cells. The shRNA for the TRAM expressing vector was generated based on the previously reported target sequence [16]. When the expression of TRAM was knocked-down (Figure 3A), NF-κB activity after IL-18 stimulation was markedly decreased relative to the negative control experiments that used scrambled shRNA (Figure 3B). This result indicates that the knock-down of TRAM expression actually impaired the IL-18 signal transduction. We confirmed the knock-down effect of this shRNA sequence for TRAM via the LPS-stimulated activation of IFN-β promoter, which is presumably due to the suppression of the MyD88-independent TLR4 pathway mediated by TRAM and TRIF (Figure 3C). The effect was similar when compared to the results obtained from a dominant negative form of TRAM (C117H). TRAM (C117H) showed an almost complete shutdown of the LPS/TLR4/IFN-β signaling in the cells (Figure 3D) and the decrease of the enhancement of NF-κB activity induced by IL-18 (Figure 3E). To further confirm the involvement of TRAM in the IL-18 signaling pathway, we evaluated the cytokine production from helper type 1 differentiated T (Th1) cells isolated from TRAM-deficient mice and MyD88-deficient mice. IL-18 did not produce IFN-γ from not only MyD88-deficient Th1 cells but also TRAM-deficient Th1 cells, significantly. And, IL-18 alone or IL-18 and IL-12 co-stimulated TRAM-deficient Th1 cells produced significantly lower IFN-γ levels than those of wild-type Th1 cells (Figure 4).


TRAM is involved in IL-18 signaling and functions as a sorting adaptor for MyD88.

Ohnishi H, Tochio H, Kato Z, Kawamoto N, Kimura T, Kubota K, Yamamoto T, Funasaka T, Nakano H, Wong RW, Shirakawa M, Kondo N - PLoS ONE (2012)

The knock-down effects of TRAM in IL-18 or LPS/TLR4 signaling.(A) RT-PCR of the TRAM mRNA knock-down in HEK293T cells by the psiRNA-TICAM-2 but not by the nonspecific scrambled sequence coded psiRNA vector. “-“ indicates cells not transfected with siRNA; “Actb” indicates the loading control (β-actin mRNA). (B–E) The effect of the knock-down of TRAM using shRNA or the dominant negative form of TRAM (C117H) for the IL-18 or LPS induced NF-κB or IFN-β-promoter luciferase activity assay. The NF-κB and IFN-β-promoter activities with IL-18 or LPS stimulation were significantly decreased in the IL-18Rβ co-transfected HEK293T cells or HEK293-hTLR4-MD2-CD14 cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369926&req=5

pone-0038423-g003: The knock-down effects of TRAM in IL-18 or LPS/TLR4 signaling.(A) RT-PCR of the TRAM mRNA knock-down in HEK293T cells by the psiRNA-TICAM-2 but not by the nonspecific scrambled sequence coded psiRNA vector. “-“ indicates cells not transfected with siRNA; “Actb” indicates the loading control (β-actin mRNA). (B–E) The effect of the knock-down of TRAM using shRNA or the dominant negative form of TRAM (C117H) for the IL-18 or LPS induced NF-κB or IFN-β-promoter luciferase activity assay. The NF-κB and IFN-β-promoter activities with IL-18 or LPS stimulation were significantly decreased in the IL-18Rβ co-transfected HEK293T cells or HEK293-hTLR4-MD2-CD14 cells.
Mentions: After obtaining the evidence that TRAM interacts with MyD88 both in vitro and in cells, we then examined the possible involvement of TRAM in IL-18 signaling. We knocked-down the endogenous TRAM expression in HEK293 cells using siRNA techniques and then performed NF-κB reporter assays for the IL-18 signal transduction in the cells. The shRNA for the TRAM expressing vector was generated based on the previously reported target sequence [16]. When the expression of TRAM was knocked-down (Figure 3A), NF-κB activity after IL-18 stimulation was markedly decreased relative to the negative control experiments that used scrambled shRNA (Figure 3B). This result indicates that the knock-down of TRAM expression actually impaired the IL-18 signal transduction. We confirmed the knock-down effect of this shRNA sequence for TRAM via the LPS-stimulated activation of IFN-β promoter, which is presumably due to the suppression of the MyD88-independent TLR4 pathway mediated by TRAM and TRIF (Figure 3C). The effect was similar when compared to the results obtained from a dominant negative form of TRAM (C117H). TRAM (C117H) showed an almost complete shutdown of the LPS/TLR4/IFN-β signaling in the cells (Figure 3D) and the decrease of the enhancement of NF-κB activity induced by IL-18 (Figure 3E). To further confirm the involvement of TRAM in the IL-18 signaling pathway, we evaluated the cytokine production from helper type 1 differentiated T (Th1) cells isolated from TRAM-deficient mice and MyD88-deficient mice. IL-18 did not produce IFN-γ from not only MyD88-deficient Th1 cells but also TRAM-deficient Th1 cells, significantly. And, IL-18 alone or IL-18 and IL-12 co-stimulated TRAM-deficient Th1 cells produced significantly lower IFN-γ levels than those of wild-type Th1 cells (Figure 4).

Bottom Line: These findings suggest that TRAM serves as the sorting adaptor for MyD88 in IL-18 signaling, which then facilitates the signal transduction.The binding sites for TRAM are located in the TIR domain of MyD88 and actually overlap with the binding sites for Mal.MyD88, the multifunctional signaling adaptor that works together with most of the TLR members and with the IL-1/IL-18 receptors, can interact with two distinct sorting adaptors, TRAM and Mal, in a conserved manner in a distinct context.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Japan. ohnishih@gifu-u.ac.jp

ABSTRACT
MyD88, a Toll/interleukin-1 receptor homology (TIR) domain-containing adaptor protein, mediates signals from the Toll-like receptors (TLR) or IL-1/IL-18 receptors to downstream kinases. In MyD88-dependent TLR4 signaling, the function of MyD88 is enhanced by another TIR domain-containing adaptor, Mal/TIRAP, which brings MyD88 to the plasma membrane and promotes its interaction with the cytosolic region of TLR4. Hence, Mal is recognized as the "sorting adaptor" for MyD88. In this study, a direct interaction between MyD88-TIR and another membrane-sorting adaptor, TRAM/TICAM-2, was demonstrated in vitro. Cell-based assays including RNA interference experiments and TRAM deficient mice revealed that the interplay between MyD88 and TRAM in cells is important in mediating IL-18 signal transduction. Live cell imaging further demonstrated the co-localized accumulation of MyD88 and TRAM in the membrane regions in HEK293 cells. These findings suggest that TRAM serves as the sorting adaptor for MyD88 in IL-18 signaling, which then facilitates the signal transduction. The binding sites for TRAM are located in the TIR domain of MyD88 and actually overlap with the binding sites for Mal. MyD88, the multifunctional signaling adaptor that works together with most of the TLR members and with the IL-1/IL-18 receptors, can interact with two distinct sorting adaptors, TRAM and Mal, in a conserved manner in a distinct context.

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