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, 20 (24), 7108-16

Glucocorticoids Inhibit MAP Kinase via Increased Expression and Decreased Degradation of MKP-1

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Glucocorticoids Inhibit MAP Kinase via Increased Expression and Decreased Degradation of MKP-1

O Kassel et al. EMBO J.

Abstract

Glucocorticoids inhibit the proinflammatory activities of transcription factors such as AP-1 and NF-kappa B as well as that of diverse cellular signaling molecules. One of these signaling molecules is the extracellular signal-regulated kinase (Erk-1/2) that controls the release of allergic mediators and the induction of proinflammatory cytokine gene expression in mast cells. The mechanism of inhibition of Erk-1/2 activity by glucocorticoids is unknown. Here we report a novel dual action of glucocorticoids for this inhibition. Glucocorticoids increase the expression of the MAP kinase phosphatase-1 (MKP-1) gene at the promoter level, and attenuate proteasomal degradation of MKP-1, which we report to be triggered by activation of mast cells. Both induction of MKP-1 expression and inhibition of its degradation are necessary for glucocorticoid-mediated inhibition of Erk-1/2 activation. In NIH-3T3 fibroblasts, although glucocorticoids up-regulate the MKP-1 level, they do not attenuate the proteasomal degradation of this protein and consequently they are unable to inhibit Erk-1/2 activity. These results identify MKP-1 as essential for glucocorticoid-mediated control of Erk-1/2 activation and unravel a novel regulatory mechanism for this anti-inflammatory drug.

Figures

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Fig. 1. Glucocorticoids inhibit Erk-1/2 activation. RBL-2H3 mast cells were treated (A) for the indicated time with dexamethasone (D, 0.1 µM) or solvent (ethanol, E), or (B) for 16 h with dexamethasone (Dex, 0.1 µM) in the presence of RU486 at the indicated concentrations. The cells were then sensitized with monoclonal anti-DNP IgE, and stimulated with DNP-BSA or activated with thapsigargin or TPA as indicated. Erk-1/2 and Elk-1 phosphorylation (p-Erk, p-Elk-1) was assessed by immunoblotting using phospho-specific antibodies. The membranes were stripped and reprobed with phosphorylation state-independent anti-Erk-2 and anti-Elk-1 antibodies. The results are representative of three different experiments.
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Fig. 2. Glucocorticoid-mediated inhibition of Erk-1/2 activation involves de novo expression of a tyrosine phosphatase. (A) RBL-2H3 mast cells were treated with dexamethasone (Dex, 0.1 µM) or solvent alone (EtOH) for 16 h in the presence or absence of cycloheximide (CHX), before sensitization with anti-DNP IgE, and activation with DNP-BSA. Phosphorylation of Erk-1/2 (p-Erk) was assessed by immunoblotting using a phospho-specific antibody. The membranes were stripped and reprobed with a phosphorylation state-independent anti-Erk-2 antibody. (B) Phosphorylated Erk-1/2 contained in lysates from IgE-sensitized and DNP-BSA-activated RBL-2H3 cells was subjected to a dephosphorylation assay. This was achieved by incubating the cellular extracts with lysates from RBL-2H3 cells treated for 16 h with dexamethasone (Dex, 0.1 µM) in the presence or absence of RU486 (1 µM) or cycloheximide (CHX). Incubation was performed in the absence (lanes 1–7) or the presence (lanes 8 and 9) of sodium orthovanadate (vanadate, 1 mM). The level of phosphorylation of Erk (p-Erk) was assessed by immunoblotting. The results are representative of three different experiments.
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Fig. 3. Glucocorticoids induce MKP-1 gene expression. RBL-2H3 mast cells (A), mouse bone marrow-derived mast cells (B) and NIH-3T3 fibroblasts (C) were treated for the indicated time with dexamethasone (Dex, 0.1 µM) or solvent alone. Total RNA was extracted and subjected to northern blotting using cDNA probes for MKP-1 and GAPDH. (D) COS-7 cells were transiently co-transfected with an MKP-1 promoter firefly luciferase construct (-1716MKP-1luc) and a Renilla luciferase construct as an internal control, together with either wild-type glucocorticoid receptor (GRwt) or two different dimerization/transactivation-deficient GR mutant [A458T or hGR(D4X)] expression vectors. Treatment with dexamethasone (Dex, 0.1 µM) or solvent alone was performed 4 h after the transfection and the cells were harvested 48 h later for luciferase activity measurements. The results are expressed as the level of MKP-1 promoter-driven firefly luciferase expression after correcting for the transfection efficiency by Renilla luciferase measurements (relative luciferase activity), and are presented as the mean ± SD of three independent experiments.
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Fig. 4. Glucocorticoid-induced increase in MKP-1 protein level correlates with inhibition of Erk-1/2 activity. RBL-2H3 mast cells were treated for the indicated time with dexamethasone (Dex, 0.1 µM) or solvent alone (ethanol) (A), for 16 h with dexamethasone (Dex) at the indicated concentration or solvent alone (ethanol, E) (B), or for 16 h with dexamethasone (Dex, 0.1 µM) in the presence of RU486 at the indicated concentration (C). The cells were then sensitized with monoclonal anti-DNP IgE, and activated with DNP-BSA [(B) and (C)]. MKP-1 protein expression was assessed by immunoblotting using a specific anti-MKP-1 antibody, and Erk-1/2 phosphorylation (p-Erk) was assessed by immunoblotting as in Figure 1. (D) Rat recombinant purified activated Erk-2 was subjected to an in vitro dephosphorylation assay, in the presence of RBL-2H3 mast cell lysates that were treated for 16 h with either dexamethasone (Dex, 0.1 µM) or solvent alone (EtOH). The dephosphorylation assay was carried out after depleting MKP-1 from the dexamethasone-treated and control cell extracts by immunoprecipitation using a specific anti-MKP-1 antibody or an isotype control antibody. The level of phosphorylation of Erk-2 (p-Erk) was assessed by immunoblotting as in Figure 1. The membranes were stripped and reprobed with an anti-MKP-1 antibody. An anti-Hsp90 antibody was used for loading control. The results are representative of two different experiments.
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Fig. 5. Dexamethasone inhibits MKP-1-targeted degradation by the proteasome in RBL-2H3 mast cells. RBL-2H3 mast cells were treated for the indicated time with dexamethasone (Dex, 0.1 µM) or solvent alone (ethanol) (A). Cells were also treated for 3 h with solvent alone (DMSO), the proteasome inhibitors MG132 or LLnL, or the cysteine proteinase inhibitor E-64d (B). The cells were sensitized with anti-DNP IgE, then stimulated with DNP-BSA [(A) and (B)]. Phosphorylation of Erk-1/2 (p-Erk) and expression of MKP-1 were assessed by immunoblotting as described in Materials and methods. The results are representative of two different experiments.
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Fig. 6. Attenuation of proteasomal degradation and induction of the expression of MKP-1 are involved in dexamethasone-mediated dephosphorylation of Erk-1/2. (A) RBL-2H3 mast cells were treated for 5 h with dexamethasone (Dex, 0.1 µM) or solvent alone (ethanol), and for 3 h with solvent alone (DMSO), the proteasome inhibitors MG132 or LLnL, or the cysteine proteinase inhibitor E-64d, prior to activation with thapsigargin (Tg). Phosphorylation of Erk-1/2 (p-Erk) and expression of MKP-1 were assessed by immunoblotting as described in Materials and methods. An anti-Hsp90 antibody was used for loading control. (B) RBL-2H3 mast cells were loaded with an anti-MKP-1 antisense morpholino-oligonucleotide or a control morpholino-oligonucleotide before treatment with either solvent alone or dexamethasone (0.1 µM) for 16 h. The cells were sensitized with anti-DNP IgE and stimulated with DNP-BSA. Phosphorylation of Erk-1/2 (p-Erk) was assessed by immunoblotting using an anti-phosphoErk antibody. The membranes were stripped and reprobed with an anti-MKP-1 antibody and with the anti-Erk-2 antibody. The results are representative of two different experiments.
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Fig. 7. Dexamethasone does not inhibit MKP-1 degradation by the proteasome in NIH-3T3 fibroblasts. NIH-3T3 fibroblasts were treated for the indicated time [(A and B)] or for 5 h (C) with dexamethasone (Dex, 0.1 µM) or solvent alone (ethanol). For the last 3 h of the treatment period, solvent alone (DMSO), the proteasome inhibitors MG132 or LLnL, or the cysteine proteinase inhibitor E-64d were added (C). The cells were then stimulated with TPA as indicated (A). Expression of MKP-1 and phosphorylation of Erk-1/2 (p-Erk) were assessed by immunoblotting as described in Materials and methods. The results are representative of two different experiments.

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