Article Figures & Data
Figures
- FIGURE 1.
Calcineurin inhibitors activate TLR signaling. A, RAW 264.7 cells were stimulated by LPS (1 μg/ml), FK506 (FK, 10 μg/ml, obtained from Fujisawa), or CsA (50 μg/ml) for 4 h. Nuclear extracts were prepared, and NF-κB DNA-binding activity was determined by EMSA. Supershift assays were performed by incubating LPS-stimulated nuclear extract with anti-p50 or anti-p65 Abs. B, RAW 264.7 cells were stimulated by LPS, FK506, or CsA for the indicated time period, and cell lysates were prepared and subjected to SDS-PAGE, followed by Western blot with indicated Abs. C, IKK in vitro kinase assay. RAW 264.7 cells were stimulated by LPS or FK506 for the indicated time period, and cell lysates were prepared and subjected to kinase assay using GST-IκΒ-α (1–54) as a substrate. Kinase activity (KA) was visualized by autoradiography, and enzyme immunoprecipitation was confirmed by immunoblotting with the corresponding anti-IKK-α or IKK-β Abs (IB). D, Peritoneal macrophages were stimulated with 0, 1, or 10 μg/ml FK506 and 0, 0.01, or 0.1 μg/ml LPS for 24 h. Culture supernatants were collected to measure TNF-α levels by ELISA. E, RAW 264.7 cells were stimulated with medium (None), LPS (0.1 μg/ml), poly(I:C) (20 μg/ml), or FK506 (10 μg/ml), and harvested after 3 h of incubation, and total RNA was prepared and subjected to RT-PCR using IFN-β (ifn-b) and IP-10 (ip-10) primers. GAPDH (gapdh) primers were used as the internal control. F, RAW 264.7 cells were stimulated with medium (None), FK506 (10 μg/ml), poly(I:C) (20 μg/ml), or LPS (0.1 μg/ml), and incubated for 6 h, and cell lysates were subjected to native PAGE and immunoblotting using anti-IRF3 Ab. G, RAW 264.7 cells were stimulated by LPS, or the indicated amount of FK506, and incubated for 30 min. Cell lysates were subjected to SDS-PAGE and immunoblotting with indicated Abs. Results shown are representative of two to four separate experiments.
- FIGURE 2.
Knockdown of calcineurin enhances the activation of TLR-mediated signaling pathway. RAW 264.7 cells were transfected with control or calcineurin-targeting siRNA, and incubated for 3 days, and cells were cultured 4 h before treatment. A, Cell lysates were prepared and analyzed by immunoblot using anti-calcineurin or GAPDH Abs. B, Cells were treated with medium (None), LPS (0.1 μg/ml), peptidoglycan (PGN, 10 μg/ml), poly(I:C) (20 μg/ml), or CpG DNA (2.5 μg/ml) for 4 h, and nuclear extracts were prepared for EMSA. To provide a comparison with effects of FK506 on levels of activated NF-κB, nontransfected cells were stimulated with FK506 for 4 h. The x-ray films of the EMSAs from nontransfected FK506-treated or siRNA-transfected cells were exposed for 4 h, whereas the EMSA film from siRNA-transfected nonstimulated cells was exposed for 16 h. C′, Cold competition of FK506-treated cell nuclear extract using unlabeled NF-κB probe. C, Cells transfected with control or calcineurin siRNA were treated with medium (None), poly(I:C), LPS, or CpG DNA for 24 h. Culture supernatants were assayed for TNF-α by ELISA. D, Cells transfected with control or calcineurin siRNA were treated with medium, LPS, or poly(I:C) for 6 h, and RNA samples were prepared for RT-PCR using IFN-β or GAPDH primers. E, The same as C, except cells were treated with medium or FK506 (10 μg/ml). Data are shown as means ± SD of triplicates. ∗, p < 0.05, and ∗∗, p < 0.01 vs control. Results shown are representative of two to three independent experiments.
- FIGURE 3.
Calcineurin inhibits the IL-1R/TLR-mediated activation of NF-κB. A, RAW 264.7 cells were transfected with pNF-κB-luc (1.5 μg), pSV-β-gal (1.5 μg), and 4.5 μg of control or constitutively active calcineurin (ΔCnA) expression vectors. After 48 h, cells were stimulated by medium (None), Pam3CSK4 (Pam3, 200 ng/ml), poly(I:C) (20 μg/ml), LPS (0.1 μg/ml), CpG DNA (2.5 μg/ml), FK506 (10 μg/ml), IL-1β (20 ng/ml), or TNF-α (10 ng/ml). Cell lysates were prepared and subjected to luciferase and β-galactosidase assays. After normalization with β-galactosidase activity, fold activation was calculated as the fold increase in luciferase activity compared with the unstimulated cells. B, 293T cells were transfected with IRAK-1-FLAG, MyD88-AU-1, and GFP-ΔCnA vectors. Cell lysates were analyzed by immunoblotting using anti-IRAK-1 Ab to examine the phosphorylation of transfected IRAK-1, and also probed with anti-AU-1 or GFP Abs. pEGFP-C1 vector was cotransfected as the control. C, RAW 264.7 cells were cotransfected with pNF-κB-luc (1.5 μg), pSV-β-gal (1.5 μg), and control, dn TRAF6, dn IRAK-1, dn MyD88, N-IRAK-1, or KA-IRAK-4 expression vectors (4.5 μg each). After 48 h, cells were stimulated by LPS (1 μg/ml) or FK506 (10 μg/ml) and incubated for 6 h. Cell lysates were prepared for luciferase and β-galactosidase assays. D, Wild-type or MyD88−/− peritoneal macrophages were treated with medium (None), CpG DNA (2.5 μg/ml), poly(I:C) (25 μg/ml), or FK506 (indicated amount in μg/ml) for 4 h. Nuclear extracts were prepared, and DNA-binding activity was analyzed by EMSA. ∗, Indicates nonspecific binding. Results shown are representative of three to four independent experiments.
- FIGURE 4.
Targets of negative regulation by calcineurin. Peritoneal macrophages from wild-type, MyD88−/−, TRIF−/−, TLR2−/−, TLR3−/−, TLR4−/−, or TLR9 mutant TLR9CpG1/CpG1 mice were A, incubated with 10 μg/ml FK506 for the indicated time period and harvested for Western blot analysis using indicated Abs, or B, incubated with medium (None), Pam3CSK4 (Pam3, 200 ng/ml), poly(I:C) (25 μg/ml), LPS (0.1 μg/ml), CpG DNA (2.5 μg/ml), or FK506 (10 μg/ml) for 24 h, as indicated in each figure. Culture supernatants were assayed for TNF-α by ELISA. Data are shown as means ± SD of triplicates. ∗, p < 0.01; ∗∗, p < 0.005; and ∗∗∗, p < 0.001 vs control. Results shown are representative of two to three independent experiments.
- FIGURE 5.
Calcineurin is associated with MyD88, TRIF, TLR2, and TLR4, not with TLR3 and TLR9. A, 293T cells were transfected with TLR4-FLAG, MyD88-AU-1, and GFP-ΔCnA or control GFP vectors. Cell lysates were immunoprecipitated with anti-AU-1 Ab and subjected to immunoblotting with indicated Abs. *, IgH. B, 293T cells were transfected with FLAG-TRIF, FLAG-IRAK-4, and GFP-ΔCnA or control GFP vectors. Cell lysates were immunoprecipitated with anti-FLAG Ab and subjected to immunoblotting with indicated Abs. C, 293T cells were transfected with FLAG-tagged TLR 2, 3, 4, or 9, and GFP-ΔCnA expression vectors. Cell lysates were immunoprecipitated with anti-FLAG Ab and subjected to immunoblotting with indicated Abs. D, RAW 264.7 cells were untreated or treated with FK506 (10 μg/ml) for 30 min, and cell lysates were immunoprecipitated with isotype or indicated Abs, and the immunoprecipitate was resolved in SDS-PAGE, followed by immunoblotting using indicated Abs. Results shown are representative of two to four independent experiments.
- FIGURE 6.
Model for the negative regulation of TLR signaling by calcineurin. A, In nonactivated (resting) macrophages, calcineurin interacts and negatively regulates the adaptor proteins MyD88 and TRIF, and TLRs expressed on the plasma membrane such as TLR2 and TLR4, not TLRs that are only expressed in endosomes (TLR3 and TLR9 have been tested), the initial components of the pathways leading to NF-κB, MAPK, and IRF3 activation. B, Calcineurin inhibitors or ligand binding to the TLRs lead to the activation of NF-κB and other transcription factors (TFs) via MyD88-dependent and MyD88-independent pathways. The filled and open symbols denote the inactive and active forms of the molecules, respectively. Endosomes are indicated as double-lined circles. See Discussion.
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