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ERK1/2 antagonize AMPK-dependent regulation of FcεRI-mediated mast cell activation and anaphylaxis  Seung-Lark Hwang, PhD, Yue Lu, PhD, Xian Li, MSc,

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Presentation on theme: "ERK1/2 antagonize AMPK-dependent regulation of FcεRI-mediated mast cell activation and anaphylaxis  Seung-Lark Hwang, PhD, Yue Lu, PhD, Xian Li, MSc,"— Presentation transcript:

1 ERK1/2 antagonize AMPK-dependent regulation of FcεRI-mediated mast cell activation and anaphylaxis 
Seung-Lark Hwang, PhD, Yue Lu, PhD, Xian Li, MSc, Yong Deuk Kim, PhD, You Sook Cho, MD, PhD, Yurndong Jahng, PhD, Jong- Keun Son, PhD, Youn Ju Lee, PhD, Wonku Kang, PhD, Yoshitaka Taketomi, PhD, Makoto Murakami, PhD, Tae Chul Moon, PhD, Hyeun Wook Chang, PhD  Journal of Allergy and Clinical Immunology  Volume 134, Issue 3, Pages e7 (September 2014) DOI: /j.jaci Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Pharmacological inhibition of ERK1/2 or activation of AMPK decreases mast cell activation. Effects of U0126 or AICAR on FcεRI-induced phosphorylation of signal molecules (15 minutes) (A), release of β-Hex (15 minutes) (B), LTC4 (15 minutes) (C), PGD2 (7 hours) (D), TNF-α (6 hours) (E), and IL-6 (6 hours) (F), and influx of Ca2+ (5 minutes) (G) in IgE/Ag-activated BMMCs (n = 3; ***P < .001, for comparison of U0126- or AICAR-treated cells with untreated cells). DNP-HSA, Dinitrophenyl-human serum albumin. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Knockdown of ERK1/2 decrease mast cell activation. BMMCs were treated with siRNA for ERK1/2 or control (mock) for 48 hours. Then, FcεRI-induced phosphorylation of signaling molecules (A), release of β-Hex (B), LTC4 (C), PGD2 (D), TNF-α (E), and IL-6 (F), and influx of Ca2+ (G) were evaluated (n = 3; **P < .01, for comparison of siRNA with mock treated cells). DNP-HSA, Dinitrophenyl-human serum albumin. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 U0126 and AICAR each inhibit IgE/Ag-induced anaphylaxis. Effects of U0126 or AICAR on IgE/Ag-induced PCA (A) and PSA (B-D). The top panel shows representative photos of ears with dye extravasation at 1 hour (Fig 3, A). Blood levels of LTC4, PGD2, and histamine were evaluated at 5 minutes (Fig 3, B). The agents were administered intraperitoneally 1 hour before Ag administration (n = 5, **P < .01, for comparison of Ag-challenged mice with vs without U0126 or AICAR). DNP-HSA, Dinitrophenyl-human serum albumin. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Knockdown of AMPKα2 increases ERK1/2 signaling in IgE/Ag-activated BMMCs. BMMCs were treated with siRNA for AMPKα2 or control (mock) for 48 hours. Then, IgE/Ag-induced phosphorylation of signaling molecules (A) and release of β-Hex (B), LTC4 (C), and PGD2 (D) were evaluated with or without U0126 or AICAR (n = 3; *P < .05, **P < .01 and ***P < .001, for comparison of siRNA with mock for each treatment). DNP-HSA, Dinitrophenyl-human serum albumin. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Knockout of AMPKα2 increases ERK1/2 signaling in IgE/Ag-activated BMMCs. A, IgE/Ag-induced PCA in WT and AMPKα2−/− mice with or without U0126 or AICAR (n = 5; #P < .01, vs without U0126 or AICAR; and **P < .01, vs WT for each treatment). The bottom panel shows representative photos of ears. B-E, IgE/Ag-induced phosphorylation of signaling molecules (Fig 5, B) and release of β-Hex (Fig 5, C), LTC4 (Fig 5, D), and PGD2 (Fig 5, E) in WT and AMPKα2−/− BMMCs with or without U0126 or AICAR (n = 3; **P < .01 and ***P < .001, for comparison of KO with WT for each treatment). DNP-HSA, Dinitrophenyl-human serum albumin; KO, knockout. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 ERK1/2 regulate AMPK subcellular distribution. A, IgE/Ag-stimulated BMMCs or nonstimulated with or without U0126 or AICAR were immunoprecipitated with anti-ERK1/2 or MEK1/2 antibody and then immunoblotted with anti-AMPK, -MEK, or -ERK1/2 antibody. The relative ratios of ERK1/2 to AMPK were determined by using scanning densitometry (n = 3, ***P < .001, for comparison of Ag-stimulated cells that had been untreated with those that had been treated with U0126 or AICAR). B, Immunoblotting of signaling molecules in the cytosol and nucleus. GAPDH and lamin B were used as respective fraction markers. DNP-HSA, Dinitrophenyl-human serum albumin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E1 MEK inhibitors dose-dependently inhibit IgE/Ag-activated mast cell signals. BMMCs were pretreated for 1 hour with various concentrations of U0126 or PD98059 and then stimulated with IgE/Ag for 15 minutes to analyze the phosphorylation of signaling molecules (A and F) and effector functions including β-Hex release (B and G), generation of LTC4 (C and H) and PGD2 (D and I), and calcium influx (E and J) (n = 3; **P < .01 and ***P < .001, vs without the compounds). Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E2 Effects of U0126 or AICAR on Fyn, Lyn, Syk, and LAT phosphorylation in IgE/Ag-stimulated BMMCs. BMMCs were sensitized with IgE with or without U0126 or AICAR pretreatment and then stimulated with DNP-HSA for 15 minutes. Immunoprecipitates of Fyn, Lyn, Syk, and LAT were analyzed by immunoblotting with antiphosphotyrosine antibody, followed by reprobing with anti-Fyn, -Lyn, -Syk, or -LAT antibody. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E3 Effects of U0126 or AICAR on IgE/Ag-activated signals in KU 812 and RBL-2H3 cells. IgE-sensitized KU 812 (A-D) and RBL-2H3 (E-H) cells with or without U0126 or AICAR were stimulated with DNP-HSA for 15 minutes and were taken for immunoblotting (Fig E3, A and E) and assays for β-HEX (Fig E3, B and F), LTC4 (C and G), and PGD2 (D and H) (n = 3; ***P < .001, vs without the compounds). Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Fig E4 Effects of LKB1 knockdown on mast cell signals. BMMCs were treated with siRNA for LKB1 or control (mock) for 48 hours. Then, IgE/Ag-induced phosphorylation of signaling molecules (A) and release of β-Hex (B), LTC4 (C), and PGD2 (D) were evaluated (n = 3; *P < .05, **P < .01, and ***P < .001, vs mock for each treatment). Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 Fig E5 Possible mechanism for the inhibitory crosstalk between ERK1/2 and AMPK in FcεRI-mediated mast cell activation. In unstimulated mast cells, LKB1 and AMPK (total and phosphorylated forms) are present in both the nuclear and cytosolic fractions. A, IgE/Ag-induced crosslinking of FcεRI induces the activation of Syk and Fyn, which activates downstream molecules such as PLCγ and MAPKs and increases calcium influx, leading to degranulation, eicosanoid generation, and cytokine expression. IgE/Ag stimulation also allows ERK1/2 and Fyn to associate with LKB1 and AMPK, which sequesters the LKB1-AMPK complex into the nucleus and thereby shuts off its inhibitory signal. B, After treatment with SU6656 (a Fyn inhibitor), U0126 (a MEK-ERK1/2 inhibitor), or AICAR (an AMPK activator), LKB1 and AMPK are redistributed into the cytosol of IgE/Ag-activated cells and prevent the ERK1/2-regulated signaling, leading to reductions in the PLCγ-mediated calcium influx and the effector functions. Red arrows indicate positive signals, whereas the blue arrow indicates the negative effect of AMPK. Dashed red arrows indicate the signaling processes that are negatively affected by AMPK. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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