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Extracellular signal–regulated kinase 5 promotes acute cellular and systemic inflammation

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Science Signaling  25 Aug 2015:
Vol. 8, Issue 391, pp. ra86
DOI: 10.1126/scisignal.aaa3206
  • Fig. 1 The kinase activities of ERK5 and MEK5 promote the inflammatory activation of ECs in vitro.

    (A to J) HMVEC-lung cells were pretreated for 1 hour with vehicle [dimethyl sulfoxide (DMSO)], 5 μM XMD8-92, 1 μM XMD17-109, or 10 μM BIX02189 before being treated for an additional 6 hours with vehicle (0.9% saline), LPS (10 μg/ml), TNF-α (100 ng/ml), FSL-1 (10 μg/ml), or IL-1β (0.1 ng/ml) while in the continuous presence of DMSO or inhibitor. The concentrations of IL-6 and IL-8 that were secreted by the cells into the culture medium were determined by enzyme-linked immunosorbent assay (ELISA). NS, not significant. *P < 0.05 and **P < 0.01 when comparing between cells treated with inflammatory stimulus in the presence or absence of inhibitor; #P < 0.05 and ##P < 0.01 when comparing between cells treated with vehicle alone and cells treated with inflammatory stimulus in the presence or absence of inhibitor. Data are means ± SD of four sample wells per group and are representative of three independent experiments.

  • Fig. 2 ERK5 and MEK5 proteins promote the inflammatory activation of ECs in vitro.

    (A to D) HMVEC-lung cells were transfected with (A and B) MAPK7-specific (to knockdown ERK5), (C and D) MAP2K5-specific (to knockdown MEK5), or (A to D) control siRNAs. Seventy-two hours later, cell lysates were analyzed by Western blotting with antibodies specific for the indicated proteins. Western blots were subjected to densitometric analysis. Bar graphs show the ratio of the abundances of (B) ERK5 and (D) MEK5 normalized to that of actin and are expressed relative to those in untreated control cells. *P < 0.05 when comparing relative densities between cells transfected with kinase-specific siRNA and those transfected with control siRNA. Data in (B) and (D) are means ± SD of the specific protein densitometry values from four independent Western blotting experiments. (E to H) HMVEC-lung cells transfected with MAPK7-specific, MAP2K5-specific, or control siRNAs were subsequently treated with vehicle (0.9% saline), LPS (10 μg/ml), or TNF-α (100 ng/ml) for 6 hours. The concentrations of IL-6 and IL-8 secreted by the cells into the culture medium were then determined by ELISA. *P < 0.05 when comparing control siRNA–transfected cells treated with inflammatory stimulus with specific siRNA–transfected cells treated with inflammatory stimulus; #P < 0.05 when comparing cells treated with vehicle alone with siRNA-transfected cells treated with inflammatory stimulus. Data in (E) to (H) are means ± SD of four sample wells per group and are representative of three independent experiments.

  • Fig. 3 ERK5 promotes the binding of neutrophils to activated ECs.

    (A and B) Confluent monolayers of HMVEC-lung cells grown in 48-well tissue culture plates were pretreated for 1 hour with vehicle (DMSO), 1 μM XMD17-109 (A), or 5 μM XMD8-92 (B) before being treated with vehicle (0.9% saline), FSL-1 (10 μg/ml), LPS (10 μg/ml), or TNF-α (100 ng/ml) for an additional 3 hours while in the continuous presence of DMSO or inhibitor. Calcein AM–labeled neutrophils were added at 3 × 105 to 6 × 105 cells per well and allowed to adhere for 20 min at 37°C before being washed with phosphate-buffered saline (PBS) to remove nonadherent cells. Pre- and postwashing fluorescence was read in a fluorescent plate reader. The relative numbers of the remaining adherent neutrophils were then calculated. *P < 0.05 and **P < 0.01 when comparing between cells treated with inflammatory stimulus in the absence or presence of inhibitor. Data are means ± SD of four (A) or six (B) sample wells per group and are representative of two independent experiments. NAA, neutrophil adhesion assay. (C) Representative images of calcein-labeled neutrophils bound to HMVEC-lung cells in the presence and absence of XMD8-92. Fluorescence images taken with a fluorescein filter set are shown in the left columns of each group, whereas translucent light microscopy images of the same field of reference are shown in the right-hand columns (under ×10 magnification). Images are representative of two independent experiments.

  • Fig. 4 ERK5 promotes the inflammatory activation of human PBMCs and monocytes in vitro.

    (A to L) PBMCs or monocytes (as indicated) were pretreated for 1 hour with vehicle (DMSO), 5 μM XMD8-92, or 1 μM XMD17-109 before being treated with vehicle (0.9% saline), FSL-1 (10 μg/ml), or LPS (10 μg/ml) for an additional 6 hours while in the continuous presence of DMSO or inhibitor. The concentrations of IL-6, IL-8, and TNF-α secreted by the cells into the culture medium were determined by ELISA. *P < 0.05 when comparing between cells treated with inflammatory stimulus in the presence or absence of inhibitor; #P < 0.05 when comparing between cells treated with vehicle alone and cells treated with inflammatory stimulus in the presence or absence of inhibitor. Data are means ± SD of four sample wells per group and are representative of three independent experiments. ag, attogram; fg, femtogram.

  • Fig. 5 ERK5 promotes the secretion of inflammatory mediators and enhances PAI-1 activity after the systemic challenge of mice with Pam3Cys or LPS.

    (A to P) Wild-type mice were treated intraperitoneally with XMD8-92 (50 mg/kg), XMD17-109 (50 mg/kg), BIX02189 (25 mg/kg), or vehicle [30% (2-hydroxypropyl)-β-cyclodextrin with or without 5% DMSO] 30 min before they were injected intravenously with Pam3Cys (2.5 mg/kg), LPS (10 mg/kg), or vehicle (0.9% saline). The plasma concentrations of IL-6, CCL2, and CCL3 and the activity of PAI-1 were quantified 2 hours after challenge. *P < 0.05 when comparing Pam3Cys- or LPS-treated mice in the presence or absence of inhibitor; #P < 0.05 when comparing untreated control mice with mice treated with Pam3Cys or LPS in the presence or absence of inhibitor. Data are means ± SD of four mice per group and are representative of two independent experiments.

  • Fig. 6 ERK5 promotes the secretion of inflammatory mediators and enhances PAI-1 activity after the systemic challenge of mice with HKSA.

    (A to D) Wild-type mice were treated intraperitoneally with XMD8-92 (50 mg/kg) or vehicle [30% (2-hydroxypropyl)-β-cyclodextrin] 30 min before being injected intravenously with HKSA (2.5 × 1010 bacteria/kg) or vehicle (0.9% saline). The plasma concentrations of IL-6, CCL2, and CCL3 and the activity of PAI-1 were quantified 2 hours after the mice were challenged. *P < 0.05 when comparing HKSA-treated mice in the presence or absence of inhibitor; #P < 0.05 when comparing untreated control mice with mice treated with HKSA in the presence or absence of inhibitor. Data are means ± SD of four mice per group and are representative of two independent experiments.

  • Fig. 7 Inhibition of ERK5 protects mice from endotoxemic mortality.

    (A and B) Wild-type mice were treated intraperitoneally with XMD8-92 (50 mg/kg) or vehicle [30% (2-hydroxypropyl)-β-cyclodextrin] 30 min before (A) or 1 hour after (B) being injected intraperitoneally with LPS [15 mg/kg (A) or 12.5 mg/kg (B)] or vehicle (0.9% saline). The survival of the mice was monitored for up to 72 hours. Data are from 8 (A) or 14 (B) mice per group from a single experiment and are representative of two independent experiments. The concentration of LPS used was chosen to achieve a 30 to 50% mortality rate (LD30 to LD50) in control mice after 72 hours. *P < 0.05 when comparing control mice with inhibitor-treated mice. Mortality data were analyzed with Kaplan-Meier curves.

  • Fig. 8 ERK5 promotes cytokine secretion in a mouse model of lung IR injury.

    (A and B) Wild-type mice were injected intraperitoneally with XMD8-92 (50 mg/kg) or vehicle [30% (2-hydroxypropyl)-β-cyclodextrin] just before being subjected to ventilated lung IR injury as described in Materials and Methods. The concentrations of IL-6 and CCL2 in the plasma of the indicted mice were quantified 1 hour after reperfusion (ischemia time, 30 min). Each point represents plasma from a single mouse. Data are from 6 to 10 mice per group from a single experiment and are representative of two independent experiments. *P < 0.05 and **P < 0.01 when comparing vehicle-treated mice with inhibitor-treated mice.

  • Table 1 Micromolar IC50 values for XMD8-92 in different ECs.
    AgonistHUVECsHMVEC-lung
    cells
    IL-6IL-8IL-6IL-8
    Pam3Cys
      10 μg/ml1.170.910.700.93
    FSL-1
      10 μg/ml3.184.681.701.55
    LPS
      10 μg/ml1.693.672.774.22
      1 μg/ml2.952.911.922.47
    TNF-α
      10 ng/ml2.014.330.932.96
    IL-1β
      0.1 ng/ml1.603.240.950.98
    Average IC502.103.291.502.19
  • Table 2 Micromolar IC50 values for XMD17-109 in different ECs.

    n.d., no data.

    AgonistHUVECsHMVEC-lung
    cells
    IL-6IL-8IL-6IL-8
    Pam3Cys
      10 μg/ml0.380.390.390.45
    FSL-1
      10 μg/ml2.021.830.820.79
    LPS
      10 μg/ml0.800.740.661.17
      1 μg/ml1.371.880.680.96
    TNF-α
      10 ng/ml0.612.630.430.59
    IL-1β
      10 ng/ml3.463.68n.d.n.d.
      1 ng/ml3.261.980.580.67
      0.1 ng/ml0.580.630.410.48
    Average IC501.561.720.570.73

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/391/ra86/DC1

    Fig. S1. Domain organization of ERK5.

    Fig. S2. XMD8-92, XMD17-109, and BIX02189 have no substantial effects on cell viability.

    Fig. S3. The ERK5 inhibitor XMD8-92 reduces the amounts of proinflammatory cytokines secreted by HMVEC-lung cells when it is added before or after they are treated with LPS.

    Fig. S4. MEK5 promotes the binding of neutrophils to activated ECs.

    Fig. S5. ERK5 promotes the secretion of CCL2 and enhances PAI-1 activity in mice 24 hours after they are challenged with LPS.

  • Supplementary Materials for:

    Extracellular signal–regulated kinase 5 promotes acute cellular and systemic inflammation

    Kevin Wilhelmsen,* Fengyun Xu, Katherine Farrar, Alphonso Tran, Samira Khakpour, Shirin Sundar, Arun Prakash, Jinhua Wang, Nathanael S. Gray, Judith Hellman

    *Corresponding author. E-mail: wilhelmsenk{at}anesthesia.ucsf.edu

    This PDF file includes:

    • Fig. S1. Domain organization of ERK5.
    • Fig. S2. XMD8-92, XMD17-109, and BIX02189 have no substantial effects on cell viability.
    • Fig. S3. The ERK5 inhibitor XMD8-92 reduces the amounts of proinflammatory cytokines secreted by HMVEC-lung cells when it is added before or after they are treated with LPS.
    • Fig. S4. MEK5 promotes the binding of neutrophils to activated ECs.
    • Fig. S5. ERK5 promotes the secretion of CCL2 and enhances PAI-1 activity in mice 24 hours after they are challenged with LPS.

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    Citation: K. Wilhelmsen, F. Xu, K. Farrar, A. Tran, S. Khakpour, S. Sundar, A. Prakash, J. Wang, N. S. Gray, J. Hellman, Extracellular signal–regulated kinase 5 promotes acute cellular and systemic inflammation. Sci. Signal. 8, ra86 (2015).

    © 2015 American Association for the Advancement of Science

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