如何改善社区安全ppt:The orphan nuclear receptor SHP acts as a neg...

The orphan nuclear receptor SHP acts as a negative regulator in inflammatory signaling triggered by Toll-like receptors
Abstract
The orphan nuclear receptor SHP (small heterodimer partner) is a transcriptional corepressor that regulates hepatic metabolic pathways. Here we identified a role for SHP as an intrinsic negative regulator of Toll-like receptor (TLR)-triggered inflammatory responses. SHP-deficient mice were more susceptible to endotoxin-induced sepsis. SHP had dual regulatory functions in a canonical transcription factor NF-κB signaling pathway, acting as both a repressor of transactivation of the NF-κB subunit p65 and an inhibitor of polyubiquitination of the adaptor TRAF6. SHP-mediated inhibition of signaling via the TLR was mimicked by macrophage-stimulating protein (MSP), a strong inducer of SHP expression, via an AMP-activated protein kinase–dependent signaling pathway. Our data identify a previously unrecognized role for SHP in the regulation of TLR signaling.
Figures at a glance
left Figure 1: SHP protects against LPS-induced lethal shock through the inhibition of inflammatory responses in vivo.
(a) Survival of Shp+/+ and Shp?/? mice (n = 14 per genotype) challenged with LPS (20 mg per kg body weight; intraperitoneally). (b) Enzyme-linked immunosorbent assay (ELISA) of TNF and IL-6 in serum from Shp+/+ and Shp?/? mice (n = 3 per group), assessed 18 h after intraperitoneal injection of various concentrations of LPS (horizontal axes; in mg per kg body weight (mg/kg)). (c) Semiquantitative RT-PCR analysis of the expression of Tnf, Il6, Il1b and Shp mRNA in spleen and liver samples from mice treated with various doses of LPS (above lanes); Actb (encoding β-actin) serves as a loading control throughout. (d) Cyclooxygenase-2 (COX-2) in lung tissue from Shp+/+ and Shp?/? mice treated with PBS or LPS (n = 3 per group). Scale bar, 50 μm. Right (below), COX-2+ cells (in ten random fields); inset, RT-PCR analysis of Shp. (e) Size of Shp+/+ and Shp?/? spleens 3 h after injection of PBS or LPS. Left margin, size (hash marks, 0.5 cm). (f,g) ELISA of serum TNF and IL-6 (f; n = 3 mice per group) and survival (g; n = 16 mice per group) of 12-week-old chimeras injected with LPS (30 mg/kg; intraperitoneally): Shp?/? mice reconstituted with Shp+/+ (Shp+/+Shp?/?) or Shp?/? (Shp?/?Shp?/?) bone marrow–derived cells, and Shp+/+ mice reconstituted with Shp?/? (Shp?/?Shp+/+) or Shp+/+ (Shp+/+Shp+/+) bone marrow–derived cells. *P < 0.05, **P < 0.01 and ***P < 0.001, compared with Shp+/+ mice treated with LPS (log-rank test (a,g) or two-tailed Student's t-test (b,d,f)). Data are from one representative of at least two (a,e,g) or three (b–d,f) independent experiments (mean and s.d. of triplicate (b,d (bottom right), f)).
Figure 2: SHP inhibits LPS-induced inflammatory responses in macrophages and splenocytes.
(a) ELISA of TNF, IL-6, IL-1β and IL-10 in culture supernatants of Shp+/+ and Shp?/? BMDMs incubated for 0–48 h with LPS (100 ng/ml). (b) Semiquantitative RT-PCR analysis of Tnf, Il6, Il1b and Il10 mRNA in Shp+/+ and Shp?/? BMDMs incubated for 0–18 h with LPS (100 ng/ml). (c) Immunoblot analysis of inducible nitric oxide synthase (iNOS) and SHP in whole-cell lysates (top) and detection of nitrite in culture supernatants (with Griess reagent; bottom) of cells as in a. (d) ELISA of TNF, IL-6, IL-1β and IL-10 in BMDMs transduced for 36 h with adenovirus encoding GFP only (Ad-GFP), SHP (Ad-SHP) or Shp-specific small interfering RNA (Ad-siSHP), at a multiplicity of infection of 10, followed by treatment for 18 h with LPS. Top, RT-PCR analysis of transduction efficiency. (e) ELISA of TNF and IL-6 in Shp+/+ and Shp?/? splenocytes and peritoneal macrophages (MΦ) stimulated for 0–48 h with LPS (100 ng/ml). *P < 0.05, **P < 0.01 and ***P < 0.001, compared with LPS-stimulated Shp+/+ cells (two-tailed Student's t-test). Data are from one representative of at least three independent experiments (mean and s.d. of triplicates (a,c (bottom), d,e)).
Figure 3: SHP is a regulator of signaling by Nod2 and RLRs but not of dectin-1 signaling.
(a) Survival of Shp+/+ and Shp?/? mice (n = 15 per genotype) challenged for 0–72 h with zymosan (0.5 mg/g; intraperitoneally). (b) ELISA of TNF and IL-6 in Shp+/+ and Shp?/? BMDMs incubated for 0–48 h with zymosan (Zym; 100 μg/ml). (c,d) ELISA of TNF and IL-6 (c) and semiquantitative RT-PCR analysis of Tnf, Il6 and Shp mRNA (d) in BMDMs transduced for 36 h with adenovirus (as inFig. 2d) before treatment for 18 h (c) or 6 h (d) with zymosan (100 μg/ml); below (d), densitometry results, presented relative to expression in unstimulated cells. (e) ELISA of TNF and IL-6 in Shp+/+ and Shp?/? BMDMs incubated for 0–48 h with curdlan (100 μg/ml). (f) Semiquantitative RT-PCR analysis of Ccl5 and Shp mRNA in Shp+/+ and Shp?/? BMDMs incubated for 0–18 h with MDP (100 ng/ml); below, densitometry (as in d). (g,h) Quantitative RT-PCR analysis of mRNA for interferon-β (Ifnb) in Shp+/+ and Shp?/? BMDMs incubated for 0–18 h with triphosphate RNA in complex with Lipofectamine 2000 (3pRNA-Lipo; 20 ng/ml; g) or poly(I:C) in complex with LyoVec (poly(I:C)-LyoVec; 100 ng/ml; h); results (by densitometry) are presented relative to expression in Shp+/+ cells at 0 h. *P < 0.05, **P < 0.01 and ***P < 0.001, compared with Shp+/+ cells (log-rank test (a) or two-tailed Student's t-test (b–d,f–h)). Data are from one representative of at least two (a) or three (b–h) independent experiments (mean and s.d. of triplicates (d,f (bottom), b,c,e,g,h)).
Figure 4: SHP regulates TLR4-mediated NF-κB signaling through an interaction between SHP and p65.
(a,b) Immunoblot analysis of phosphorylated (p-) IKKα-IKKβ and total IκBα and SHP in Shp+/+ and Shp?/? BMDMs stimulated with LPS (100 ng/ml); below, densitometry. (b) Immunofluorescence microscopy of p65 (green) in Shp+/+ and Shp?/? BMDMs stimulated with LPS (100 ng/ml); nuclei are stained with the DNA-intercalating dye DAPI (blue). Scale bar, 50 μm. Right, quantification of DAPI+ or p65+ pixels in the cytoplasm and nucleus (numbers correspond to time of LPS incubation), assessing the nuclear translocation of p65. FITC, fluorescein isothiocyanate. (c) Luciferase assay of Tnf promoter activity in BMDMs transduced with adenovirus (as inFig. 2d), plus adenovirus carrying a TNF luciferase reporter construct, then stimulated for 6 h with LPS; results are presented relative to activity in unstimulated cells. Above, RT-PCR analysis of transduction efficiency. (d,e) Immunoprecipitation (IP) of p65, p50 or immunoglobulin G (IgG; d) or of SHP (e) from RAW264.7 cells transduced with adenovirus (as inFig. 2d) before stimulation for 1 h with LPS, followed by PCR with primers specific for the Tnf promoter (d) or immunoblot analysis (IB) with antibody to p65 or SHP (e). Input (d, bottom), PCR analysis of DNA without immunoprecipitation; TNF-p65 (top), p65-binding region of the Tnf promoter. (f) Confocal microsopy of NF-κB (green) and SHP (red) in BMDMs expressing endogenous SHP (Endo SHP) or overexpressing SHP via adenovirus encoding SHP (Overexp SHP), assessed after stimulation for 0, 15 or 30 min with LPS. Scale bar, 10 μm. Right, dual-color pixel analysis of the colocalization of NF-κB and SHP. (g) Quantitative analysis of the colocalization coefficients in f. *P < 0.05 and **P < 0.01, compared with Shp+/+ cell cultures (two-tailed Student's t-test). Data are from one representative of at least three independent experiments (mean and s.d. of triplicates (a (bottom), c,g)).
Figure 5: SHP interacts with TRAF6 to negatively modulate its ubiquitination.
(a) Immunoblot analysis of IRAK1 and SHP in Shp+/+ and Shp?/? BMDMs stimulated for 0–60 min with LPS (100 ng/ml). (b,c) Immunoprecipitation of endogenous TRAF6 from lysates of LPS-treated Shp+/+ and Shp?/? BMDMs (b) or RAW264.7 cells transduced with adenovirus encoding SHP (c), followed by immunoblot analysis with antibody to TRAF6, ubiquitin (Ub) or SHP. MOI, multiplicity of infection. Below, immunoblot analysis without immunoprecipitation (loading control throughout). (d) Immunoprecipitation of endogenous SHP from lysates of LPS-stimulated RAW264.7 cells, followed by immunoblot analysis with antibody to TRAF6, TRAF2 or SHP. (e) Microscopy of BMDMs left unstimulated (US) or stimulated for 30 min with LPS, then stained with DAPI (blue) and immunolabeled with antibody to SHP (conjugated to the red fluorescent dye TRITC) or antibody to TRAF6 (conjugated to the green fluorescent dye Alexa Fluor 488 (Alexa488)). Scale bar, 20 μm. Below, quantification of fluorescence intensity; distance (horizontal axis) is relative to the white bars in the far right images above. (f) Immunoprecipitation of SHP from LPS-stimulated RAW264.7 cells (subjected to subcellular fractionation), followed by immunoblot analysis with antibody to TRAF6 or p65. (g) Immunoprecipitation (with antibody to hemagglutinin) of Flag-tagged TRAF6 (Flag-TRAF6) together with hemagglutinin-tagged SHP (HA-SHP) from lysates of HEK293 human epithelial T cells left untransfected (?) or transfected with plasmid encoding full-length Flag-tagged TRAF6 or TRAF6 deletion mutants consisting of amino acids 132–530 or 212–530, followed by immunoblot analysis with antibody to Flag or hemagglutinin. WCL, immunoblot analysis of whole-cell lysates without immunoprecipitation. (h) Immunoprecipitation (with antibody to Flag) of proteins from HEK293 cells overexpressing Myc-tagged SHP (Myc-SHP) or mock vector (Myc-Mock), mock transfected (Mock) or transfected with plasmid encoding Flag-tagged wild-type TRAF6 (WT) or TRAF6 with deletion of the RING domain (ΔR), with or without plasmid encoding hemagglutinin-tagged ubiquitin (HA-Ub), followed by immunoblot analysis with antibody to Flag or hemagglutinin. (i) Quantitative RT-PCR analysis of Tnf and Il6 mRNA in RAW264.7 cells transfected with empty vector control (Mock) or TRAF6 with deletion of the RING domain, followed by transduction with adenovirus encoding GFP only or SHP and stimulation for 6 h with LPS; results are presented relative to expression in unstimulated cells. Data are from one representative of at least three independent experiments (mean and s.d. of triplicates in i).
Figure 6: TLR4 activation induces SHP expression in macrophages via Ca2+-dependent activation of AMPK.
(a,b) RT-PCR analysis of Shp mRNA (a) and immunoblot analysis of SHP (b) in BMDMs stimulated with LPS (100 ng/ml). (c) Immunoblot analysis of phosphorylated AMPKα and ACC in BMDMs stimulated with LPS. Below, densitometry. (d) Microscopy of Ca2+ in THP-1 cells left unstimulated (US) or stimulated with LPS (LPS), assessed with the fluorescent Ca2+ indicator Fluo-2 AM (top), and kinetics of Ca2+ influx in THP-1 cells stimulated with LPS at 100 s and treated with ATP at 1,000 s, in images captured at intervals of 5 s (bottom). Original magnification (top), ×400. (e) Immunoblot analysis of phosphorylated AMPKα and ACC and total SHP in BMDMs left unstimulated (US), treated with dimethyl sulfoxide (D) or stimulated for 4 h (AMPKα and ACC analysis) or 36 h (SHP analysis) with LPS, plus increasing concentrations (wedges) of the CaMK inhibitor KN93 (5, 10 or 25 μM), the calcium-specific chelator BAPTA-AM (BAPTA; 5, 10 or 25 μM) or the AMPK inhibitor compound C (CompC; 5, 10 or 25 μM). (f) RT-PCR analysis of BMDMs left unstimulated or stimulated for 24 h with LPS, then treated as in e. (g) Immunoblot analysis of lysates of BMDMs transduced with lentivirus expressing nonspecific shRNA (shNS) or shRNA specific for CaMKKβ (shCaMKKβ) or AMPK (shAMPK), then cultured for 36 h in the presence or absence of LPS, probed with antibody to SHP, CaMKKβ or AMPK. (h) Immunoblot analysis of SHP and USF1 in BMDMs transduced with lentivirus expressing nonspecific shRNA or shRNA specific for USF1 (shUSFI), then incubated for 36 h with LPS with or without compound C. Data are from one representative of at least three independent experiments.
Figure 7: MSP-induced SHP regulates TLR4-mediated proinflammatory signaling through the activation of an LKB1-dependent AMPK pathway.
(a) RT-PCR analysis of Shp mRNA in BMDMs treated for 0–24 h with MSP (100 ng/ml). Below, densitometry. (b) RT-PCR analysis of Shp mRNA in BMDMs transduced for 36 h with adenovirus encoding GFP only (Ad-GFP), dominant negative AMPK (Ad-DN) or constitutively active AMPK (Ad-CA; multiplicity of infection, 10) and treated for 6 h with MSP (100 ng/ml). Below, densitometry (as inFig 3d). (c) Immunoblot analysis of phosphorylated AMPKα and total LKB1 and SHP in BMDMs transduced with lentivirus encoding nonspecific shRNA or shRNA specific for LKB1 (shLKB1; multiplicity of infection, 10) and treated for 4 h with MSP (100 ng/ml). (d) ELISA of TNF, IL-10, IL-6 and IL-1β in Shp+/+ and Shp?/? BMDMs pretreated for 4 h with MSP (100 ng/ml), followed by incubation for 18 h with LPS (100 ng/ml). (e) Immunoprecipitation of endogenous TRAF6 from BMDMs pretreated with MSP as in d and incubated for 30 min with LPS (100 ng/ml), followed by immunoblot analysis with antibody to ubiquitin or TRAF6. (f) Immunoblot analysis of NF-κB and phosphorylated mitogen-activated protein kinases (p-p44/42 and p-p38) in BMDMs pretreated with MSP as in d and stimulated for 0–120 min with LPS (100 ng/ml). (g) Confocal microscopy of p65 in cells as in f, stained with antibody to p65 and counterstained with DAPI. Scale bars, 50 μm. Right, analysis of the nuclear translocation of p65 (as inFig. 4b). (h) Immunoprecipitation of endogenous SHP from RAW264.7 cells pretreated as in d and incubated for 30 min with LPS (100 ng/ml), followed by immunoblot analysis with antibody to p65 or SHP. Data are from one representative of at least three independent experiments (mean and s.d. of triplicates (a,b (bottom) and d)).
right