Research ArticleInflammation

MerTK signaling in macrophages promotes the synthesis of inflammation resolution mediators by suppressing CaMKII activity

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Science Signaling  25 Sep 2018:
Vol. 11, Issue 549, eaar3721
DOI: 10.1126/scisignal.aar3721
  • Fig. 1 MerTK is required for Gas6-mediated suppression of the CaMKII–p38–MK2–5-LOX–SPM signaling pathway in human macrophages.

    (A to C) Human monocyte–derived macrophages were transfected with either scrambled siRNA or MERTK-specific siRNA. After 72 hours, the cells were incubated for 7 hours with control medium (Con) or 10 nM Gas6-conditioned medium. (A) Left: The macrophages were lysed and analyzed by Western blotting with antibodies specific for phosphorylated (p-) and total MerTK, CaMKII, p38, MK2, and 5-LOX. β-Actin served as the loading control. Blots show samples from three different donors. Right: Bar graphs show the ratio of the amounts of phosphorylated protein to total protein, which was quantified by densitometry with ImageJ software. Data are means ± SEM of three different donors. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control medium by one-way analysis of variance (ANOVA) with post hoc t tests for group comparisons; n.s., not significant. (B) Medium from the indicated macrophages was assayed by ELISA to detect iLXA4 and iRvD1. (C) During the last hour of Gas6 treatment, macrophages were incubated with or without 10 μM AA to induce leukotriene production. The amount of iLTB4 in the culture medium was then analyzed by ELISA. Data in (B) and (C) are means ± SEM of three to six donors. **P < 0.01 and ***P < 0.001 versus control medium by one-way ANOVA with post hoc t tests for group comparisons.

  • Fig. 2 Gas6 inactivates CaMKII by inducing the expression of ATP2A2 mRNA and SERCA2 protein and reducing the concentration of cytosolic Ca2+.

    (A) Human macrophages were transfected with scrambled siRNA or MERTK-specific siRNA and were incubated with control or Gas6-conditioned medium for the indicated times. The cells were then loaded with the Ca2+ probe Fluo3-AM, and the cytosolic Ca2+ concentration was determined by flow cytometry. Data are means ± SEM of three different donors. **P < 0.01 versus scrambled siRNA by unpaired t test. (B) Macrophages transfected with scrambled RNA or MERTK siRNA were incubated with control or Gas6-conditioned medium for 7 hours. The relative abundances of ATP2A2 mRNA (top) and SERCA2 protein (bottom) were assayed by real-time quantitative polymerase chain reaction (qPCR) and Western blotting analysis, respectively. (C) Macrophages were pretreated with 2 μM thapsigargin (Thaps) for 5 min and then incubated with control or Gas6-conditioned medium for 7 hours. Left: Cells were lysed and analyzed by Western blotting with antibodies specific for phosphorylated (p-) and total CaMKII, p38, MK2, and 5-LOX. β-Actin served as the loading control. Blots show samples from three different donors. Right: Bar graphs show the ratios of the amounts of phosphorylated protein to total protein, which were quantified by densitometry with ImageJ software. Data in the bar graphs in (B) and (C) are means ± SEM of three different donors. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control medium by one-way ANOVA with post hoc t tests for group comparisons.

  • Fig. 3 Gas6-MerTK signaling induces the expression of ATP2A2 mRNA and SERCA2 protein in human macrophages by activating ERK.

    (A) Human macrophages transfected with scrambled siRNA or MERTK-specific siRNA were incubated with control or Gas6-conditioned medium for 30 min, lysed, and then analyzed by Western blotting with antibodies specific for MerTK, p-ERK1/2, ERK1/2, and β-actin (loading control). Left: Western blots show samples from three different donors. Right: Densitometric analysis of the ratio of the abundances of p-ERK1/2 to total ERK1/2. (B) Macrophages were pretreated with vehicle or 10 μM U0126 for 30 min and then incubated with control or Gas6-conditioned medium for 7 hours. The cells were then analyzed by real-time qPCR and Western blotting analysis, respectively, to determine the relative abundances of ATP2A2 mRNA (left) and SERCA2 protein (right). Western blots show samples from three different donors. (C) Macrophages were treated as described in (B) and then were analyzed by Western blotting with antibodies specific for phosphorylated (p-) and total CaMKII, p38, MK2, and 5-LOX. β-Actin served as the loading control. Left: Western blots show samples from three different donors. Right: Bar graphs show the ratios of the indicated phosphorylated protein to total protein, which were quantified by densitometry with ImageJ software. (D) Macrophages were treated as described in (B), after which the culture medium was subjected to ELISAs to quantify iLXA4 or iLTB4. For the macrophages used for the iLTB4 assay, 10 μM AA was added to the medium during the last hour of Gas6 treatment. For all bar graphs, data are means ± SEM of three different donors. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA with post hoc t tests for group comparisons.

  • Fig. 4 Specific tyrosine residues in the cytoplasmic tail of MerTK are required for the activation of ERK and CaMKII.

    (A) HEK 293 cells were transfected with various CDMer plasmids encoding WT MerTK, the Y825F, Y867F, and Y924F mutants, and the KD mutant. Forty-eight hours later, the cells subjected to the transfection procedure without plasmids served as control (“Mock”). The cells were lysed and analyzed by Western blotting with antibodies specific for p-ERK1/2, ERK1/2, and β-actin. The bar graph shows the ratios of phosphorylated ERK1/2 to total ERK1/2, which were quantified by densitometry with ImageJ software. Data are means ± SEM of three independent experiments. ***P < 0.001 versus WT by one-way ANOVA with post hoc t tests for group comparisons. (B) Bone marrow–derived macrophages (BMDMs) from Mertk−/− mice were transduced with pMSCV–human MERTK (WT) or pMSCV–human MERTK Y872F. Seventy-two hours later, the cells were then incubated with control or Gas6-conditioned medium for 30 min (left) or 7 hours (right), which was followed by flow cytometric quantification of p-ERK1/2 or p-CaMKII, respectively. Data are means ± SEM of three different mice. **P < 0.01 and ***P < 0.001 versus control medium by one-way ANOVA with post hoc t tests for group comparisons. MFI, mean fluorescence intensity.

  • Fig. 5 ERK inhibition suppresses inflammation resolution in zymosan-induced peritonitis in mice.

    (A) WT mice were co-injected intraperitoneally with 0.1 mg of zymosan and either U0126 (25 μg/kg) or an equal volume of DMSO as the vehicle control. Peritoneal exudates were collected by lavage with 3 ml of cold PBS at the indicated times, and leukocytes and exudate fluid were separated by centrifugation at 500g for 10 min. Total leukocyte number in the exudate was counted with a hemocytometer, and the percentage of Ly6G+ neutrophils was determined by flow cytometry. Neutrophil number was calculated as total leukocytes × percentage of neutrophils. Resolution intervals (Ri) were calculated as previously described (30). Data are means ± SEM of four mice per group. **P < 0.01 and ***P < 0.001 versus vehicle-treated mice by unpaired t test. (B to D) WT or Mertk−/− mice were injected intraperitoneally with zymosan, which was followed 64 hours later by intraperitoneal injection with U0126 or DMSO. After an additional 8 hours, peritoneal exudates were collected. (B) Neutrophil number was calculated as described in (A). (C) Peritoneal leukocytes were stained with phycoerythrin (PE)–conjugated anti-F4/80 antibody, which was followed by fixation and permeabilization. Permeabilized cells were stained with anti–p-CaMKII antibody and then an Alexa Fluor 647–conjugated secondary antibody. The MFI of p-CaMKII in the macrophages was quantified by flow cytometry. (D) Exudate iLXA4 was assayed by ELISA. For (B) to (D), data are means ± SEM of four mice per group. *P < 0.05 and ***P < 0.001 versus vehicle-treated WT mice by one-way ANOVA with post hoc t tests for group comparisons.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/549/eaar3721/DC1

    Fig. S1. Validation of the MK2 and 5-LOX antibodies with siRNAs; MerTK deletion does not alter CaMKII activity in the absence of Gas6; and protein S can suppress the CaMKII pathway.

    Fig. S2. Gas6 reduces cytosolic Ca2+ in a MerTK-dependent manner, and BAPTA-AM suppresses CaMKII–p38–MK2–5-LOX signaling.

    Fig. S3. Further studies related to MerTK ligands and demonstration that Axl does not mediate Gas6-induced ATP2A2 expression.

    Fig. S4. Protein S and apoptotic cells activate ERK1/2 in macrophages, and ERK1/2 is required for Gas6-mediated suppression of CaMKII–p38–MK2–5-LOX signaling.

    Fig. S5. Detection of the cell surface expression of CDMer proteins in transfected HEK 293 cells.

    Fig. S6. Tyr872 in the cytoplasmic tail of human MerTK is required for the activation of ERK1/2 and CaMKII.

    Fig. S7. Summary scheme of MerTK-mediated resolution signaling.

  • This PDF file includes:

    • Fig. S1. Validation of the MK2 and 5-LOX antibodies with siRNAs; MerTK deletion does not alter CaMKII activity in the absence of Gas6; and protein S can suppress the CaMKII pathway.
    • Fig. S2. Gas6 reduces cytosolic Ca2+ in a MerTK-dependent manner, and BAPTA-AM suppresses CaMKII–p38–MK2–5-LOX signaling.
    • Fig. S3. Further studies related to MerTK ligands and demonstration that Axl does not mediate Gas6-induced ATP2A2 expression.
    • Fig. S4. Protein S and apoptotic cells activate ERK1/2 in macrophages, and ERK1/2 is required for Gas6-mediated suppression of CaMKII–p38–MK2–5-LOX signaling.
    • Fig. S5. Detection of the cell surface expression of CDMer proteins in transfected HEK 293 cells.
    • Fig. S6. Tyr872 in the cytoplasmic tail of human MerTK is required for the activation of ERK1/2 and CaMKII.
    • Fig. S7. Summary scheme of MerTK-mediated resolution signaling.

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