1. CaMKII promotes TLR-triggered proinflammatory cytokine and type I interferon production by directly binding and activating TAK1 and IRF3 in macrophages
by Xingguang Liu, Ming Yao, Nan Li, Chunmei Wang, Yuanyuan Zheng, and Xuetao Cao
Blood
Volume 112(13):
December 15, 2008
©2008 by American Society of Hematology

2. TLR ligands induce intracellular Ca2+ release and CaMKII activation in macrophages.
TLR ligands induce intracellular Ca2+ release and CaMKII activation in macrophages. (A) RAW264.7 cells were loaded with Fluo 3/AM and stimulated with 0.1 μg/mL LPS in the absence or presence of 1.5 mM ethyleneglycoltetraacetic acid or 6 μM BAPTA-AM, then imaged by Leica TCS SP2 confocal microscopy under a 10×/0.40 CS objective lens at 10-second intervals. The basal (left panel) and peak (middle panel) fluorescence is displayed. Graphs (right panel) showed changes in mean fluorescence intensity from 15 cells per microscopic field over time. Data are representative of 3 independent experiments (original magnification ×100). L indicates LPS. (B) Cell extracts of TLR ligand–stimulated RAW264.7 cells were immunoprecipitated with anti–CaMKII-α antibody. The immunoprecipitates were subjected to CaMKII activity assay with autocamtide-2 as substrate or immunoblotted with anti–CaMKII-α antibody. Data are shown as mean plus or minus SD of 3 independent experiments. *P < .05, **P < .01 vs unstimulated cells.
Xingguang Liu et al. Blood 2008;112:
©2008 by American Society of Hematology

3. Blockade of CaMKII activation with KN62 or silencing of CaMKII expression attenuates TLR4-activated proinflammatory cytokine and type I IFN production in macrophages.
Blockade of CaMKII activation with KN62 or silencing of CaMKII expression attenuates TLR4-activated proinflammatory cytokine and type I IFN production in macrophages. (A) Mouse peritoneal macrophages (4 × 105) were pretreated with 15 μM KN62 for 30 minutes followed by stimulation with 0.1 μg/mL LPS for the indicated time. The production of IL-6, TNF-α, or IFN-β was measured by ELISA, and IFN-4α mRNA expression was measured by Q-PCR. (B) (Top) RAW264.7 cells were transfected with control small RNA (Ctrl) or CaMKII-α siRNA 1. After 48 hours, CaMKII-α and β-actin expression in the cells was detected by immunoblot. (Bottom) CaMKII-α–silenced RAW264.7 cells were transfected with CaMKII-α full-length expression vector. After 36 hours, CaMKII-α and β-actin expression in the cells was detected by immunoblot. Similar results were obtained in 3 independent experiments. (C) Mouse peritoneal macrophages (4 × 105) were transfected with control small RNA (Ctrl) or CaMKII-α siRNA1. After 48 hours, the cells were stimulated with 0.1 μg/mL LPS for the indicated time. IL-6, TNF-α, or IFN-β in the supernatants was measured by ELISA, and IFN-4α mRNA expression was measured by Q-PCR. (D) RAW264.7 cells (1.5 × 105) were transfected with control small RNA (Ctrl) or CaMKII-α siRNA1. After 36 hours, the cells were transfected with full-length CaMKII-α plasmid. Thirty-six hours later, the cells were stimulated with 0.1 μg/mL LPS for the indicated time. IL-6 and IFN-β in the supernatants were measured by ELISA. Data are shown as mean plus or minus SD of 3 independent experiments. **P < .01.
Xingguang Liu et al. Blood 2008;112:
©2008 by American Society of Hematology

4. Overexpression of constitutively active CaMKII enhances TLR-triggered proinflammatory cytokine and IFN-β production in macrophages.
Overexpression of constitutively active CaMKII enhances TLR-triggered proinflammatory cytokine and IFN-β production in macrophages. RAW264.7 cells (1.5 × 105) (A,C,D) were transiently transfected with constitutively active CaMKII plasmid (CaMKII290). Mouse peritoneal macrophages (4 × 105) (B) were nucleofected with CaMKII290 using Amaxa Nucleofector II Biosystems. After 36 hours, the cells were stimulated with 0.1 μg/mL LPS (A,B), 0.3 μM CpG ODN (C), or 10 μg/mL poly(I:C) (D), respectively, for the indicated time. IL-6, TNF-α, or IFN-β in the supernatants was detected by ELISA. Data are shown as mean plus or minus SD of 3 independent experiments. HEK293 cells were cotransfected with 50 ng of MyD88 (E) or TRIF (F,G) expressing plasmid, 50 ng of TNF-α (E,G), or IFN-β (F) luciferase reporter plasmid, 10 ng of pTK-Renilla luciferase, together with indicated amount of CaMKII290 expressing plasmid. Total amounts of plasmid DNA were equalized using empty control vector. After 24 hours of culture, luciferase activity was measured and normalized by Renilla luciferase activity. The expression of CaMKII290 in HEK293 cells was immunoblotted with anti-flag antibody (E). Data are shown as mean plus or minus SD (n = 5) of one typical experiment from 3 independent experiments with similar results. *P < .05; **P < .01.
Xingguang Liu et al. Blood 2008;112:
©2008 by American Society of Hematology

5. Activation of CaMKII enhances MAPK, NF-κB, and IRF3 activation in TLR-triggered macrophages.
Activation of CaMKII enhances MAPK, NF-κB, and IRF3 activation in TLR-triggered macrophages. (A) RAW264.7 cells were pretreated with 15 μM KN62 (left panel) for 30 minutes or transfected with CaMKII290 plasmid and then cultured for 36 hours (right panel). The cells were stimulated with 0.1 μg/mL LPS for the indicated time. Phospho-ERK, JNK, p38, IκBα, and total ERK were detected by immunoblot. (B) RAW264.7 cells were transfected with 100 ng of pGL3.5XκB-luciferase, 10 ng of pTK–Renilla luciferase, together without (left panel) or with indicated amount of CaMKII290 plasmid (right panel). After 36 hours, the cells were stimulated with 0.1 μg/mL LPS for 6 hours. Luciferase activity was measured and normalized by Renilla luciferase activity. (C,D) RAW264.7 cells were treated as described in panel A; the whole cell extracts (C) or nuclear extracts (D) were prepared. Phospho- (C) or total-IRF3 (D) was detected by immunoblot. Data are representative of 3 separate experiments. (E) RAW264.7 cells were transfected with 100 ng of IRF3 luciferase reporter plasmids (80 ng of Gal4 luciferase reporter plasmid, 20 ng of Gal4-IRF3–expressing plasmid), 10 ng of pTK-Renilla luciferase, together with indicated amount of CaMKII290 plasmid. After 36 hours, the cells were stimulated with 0.1 μg/mL LPS for 6 hours. Luciferase activity was measured. Data are shown as mean plus or minus SD (n = 5) of one typical result from 3 independent experiments with similar results. *P < .05; **P < .01.
Xingguang Liu et al. Blood 2008;112:
©2008 by American Society of Hematology

6. CaMKII directly binds, phosphorylates, and activates TAK1.
CaMKII directly binds, phosphorylates, and activates TAK1. (A,B) RAW264.7 cells were stimulated with 0.1 μg/mL LPS for the indicated time. Equal amount cell lysates were immunoprecipitated with CaMKII-α (A) or TAK1 (B) antibody and then immunoblotted (IB) with CaMKII-α and TAK1 antibody. (C) HEK293 cells were transfected with TAK1-HA and wt or mutant CaMKII-α–flag construct as indicated. After 24 hours, cell extracts were immunoprecipitated with anti-flag antibody and then immunoblotted with anti-HA and anti-flag antibody. C indicates catalytic domain (amino acids 1-260); R, regulatory domain (amino acids ); A, association domain (amino acids ). (D) GST pull-down assays were performed with recombinant GST-tagged CaMKII-α and cell extracts of RAW264.7 cells. (E) One microgram of recombinant TAK1 protein was incubated with recombinant active CaMKII-α at 30°C for 30 minutes. Samples were separated by SDS-PAGE followed by autoradiography. (F) MBP was added into the reaction mixture as in panel E followed by incubation for another 20 minutes. Samples were analyzed by immunoblotting with anti–phospho-MBP antibody. Data are representative of 3 independent experiments.
Xingguang Liu et al. Blood 2008;112:
©2008 by American Society of Hematology

7. CaMKII directly binds, phosphorylates IRF3, and enhances IRF3-activated IFN-β expression.
CaMKII directly binds, phosphorylates IRF3, and enhances IRF3-activated IFN-β expression. (A,B) RAW264.7 cells were stimulated with 0.1 μg/mL LPS for the indicated time. Equal amount cell lysates were immunoprecipitated with CaMKII-α (A) or IRF3 (B) antibody and then detected with CaMKII-α and IRF3 antibody. (C) HEK293 cells were transfected with IRF3-HA and wt or mutant CaMKII-α–flag construct as described in Figure 5C. After 24 hours, cell extracts were immunoprecipitated with anti-flag antibody and then detected with anti-HA and anti-flag antibody. (D) CaMKII-α–flag construct together with HA-tagged IRF3, IRF3N140, or IRF3C141 plasmid were transfected into HEK293 cells. After 24 hours, IRF3 truncates were immunoprecipitated with HA-specific antibody. Precipitated proteins were detected by immunoblot. (E) GST pull-down assays were performed with GST-tagged CaMKII-α and RAW264.7 cell lysates. (F) wt or mutant GST-IRF were used as substrates of recombinant active CaMKII-α. The incorporation of 32P in the IRF was visualized by autoradiography after SDS-PAGE. Residues are as follows: 2A, S385A, S386A; 5A, S396A, S398A, S402A, T404A, S405A; 7A, S385A, S386A, S396A, S398A, S402A, T404A, S405A. Data are representative of 3 independent experiments. (G) HEK293 cells were transfected with 100 ng of IRF3-expressing plasmid, 50 ng of IFN-β luciferase reporter plasmid, 10 ng of pTK-Renilla luciferase, together with indicated amount of CaMKII290 plasmid. After 24 hours, luciferase activity was measured and normalized by Renilla luciferase activity. Data are shown as mean plus or minus SD (n = 5) of 1 typical experiment from 3 independent experiments with similar results. *P < .05; **P < .01.
Xingguang Liu et al. Blood 2008;112:
©2008 by American Society of Hematology

8. Blockade of CaMKII activation by KN62 protects mice from endotoxin shock after lethal LPS challenge.
Blockade of CaMKII activation by KN62 protects mice from endotoxin shock after lethal LPS challenge. (A-C) Sex- and age-matched mice were injected intraperitoneally with 25-mg/kg doses of KN62 (the working concentration of KN62 is 7.2 g/L) (n = 8) or equal volume of dimethyl sulfoxide (n = 8) 30 minutes before intraperitoneal administration with 10 mg/kg body weight of LPS. Serum samples were obtained at 1.5 hours after LPS injection. Serum IL-6 (A), TNF-α (B), and IFN-β (C) were quantified by ELISA. Data represent mean plus or minus SD. *P < .05. Three experiments were performed with similar results. (D) Survival curve of mice (n = 8 per group) treated as described in panels A to C. The survival of the LPS-challenged mice was monitored for 7 days. *P < .05. Similar results were obtained in 3 independent experiments.
Xingguang Liu et al. Blood 2008;112:
©2008 by American Society of Hematology