p38δ controls Mitogen- and Stress-activated Kinase-1 (MSK1) function in response to toll-like receptor activation in macrophages

Abstract

Mitogen- and Stress-activated Kinase (MSK) 1 is a nuclear protein, activated by p38α Mitogen-Activated Kinase (MAPK) and extracellular signal-regulated kinase (ERK1/2), that modulate the production of certain cytokines in macrophages. Using knockout cells and specific kinase inhibitors, we show that, besides p38α and ERK1/2, another p38MAPK, p38δ, mediates MSK phosphorylation and activation, in LPS-stimulated macrophages. Additionally, recombinant MSK1 was phosphorylated and activated by recombinant p38δ, to the same extent than by p38α, in in vitro experiments. Moreover, the phosphorylation of the transcription factors CREB and ATF1, that are MSK physiological substrates, and the expression of the CREB-dependent gene encoding DUSP1, were impaired in p38δ-deficient macrophages. Also, the transcription of IL-1Ra mRNA, that is MSK-dependent, was reduced. Our results indicate that MSK activation can be one possible mechanism by which p38δ regulates the production of a variety of inflammatory molecules involved in immune innate response.

Keywords: MAPK; MSK1; macrophages; p38δ/p38γ; phosphorylation.

FIGURE 1

MSK1 phosphorylation in impaired in p38δ^−/− macrophages. (A) Schematic representation of the TLR4 signalling pathways involved in MAPK and NFκB pathway activation. TLR4 stimulation by LPS triggers the activation of TAK1-IKK-TPL2 via MyD88. p38γ and p38δ regulate TPL2 steady-state levels, which is in a complex with ABIN-2 and p105 (3, 8). The kinases blocked by the indicated inhibitors are shown. (B,C) BMDM from WT, p38γ^−/−, p38δ^−/− or p38γ/δ^−/− mice were stimulated with 100 ng/mL LPS for 30 min. Cell lysates were immunoblotted with the indicated antibodies. Representative immunoblots from three independent experiments in duplicate are shown. (D) BMDM from WT or p38δ^−/− mice were stimulated with 250 ng/mL CpG-ODN for the times indicated. Cell lysates were immunoblotted with the indicated antibodies. Representative immunoblots from three different experiments in duplicate are shown. Molecular weights are indicated at the side of the blots.

FIGURE 2

MSK1 phosphorylation in blocked by ERK1/2 and p38MAPK inhibitors in macrophages. (A) Raw 264.7 cells were incubated for 1 h with or without 2 µM PD184352, 10 μM SB203580, 3 µM JNK-IN-8 (to inhibit JNKs) or 0.1 µM (to inhibit p38α/β), 1 µM (to inhibit p38γ) or 10 µM (to inhibit p38δ) BIRB0796 and then stimulated with LPS as in (Figures 1B, C). Cell lysates were immunoblotted with the indicated antibodies. Representative immunoblots from two independent experiments in duplicate are shown. (B) MSK1 band from panel (A) were quantified using the Fiji program. Data show mean ± SEM from two experiments in duplicate. **p ≤ 0.01; ***p ≤ 0.001. (C) WT BMDM were incubated for 1 h with or without 2 µM PD184352, 10 μM SB203580, 3 µM JNK-IN-8 (to inhibit JNKs) or 0.1 µM (to inhibit p38α/β), 1 µM (to inhibit p38α/β and p38γ) or 10 µM (to inhibit p38α/β, p38γ and p38δ) BIRB0796 and then stimulated with 100 ng/mL LPS for 60 min. Cell lysates were immunoblotted with the indicated antibodies. Representative immunoblots from two independent experiments in duplicate are shown. (D) MSK1 band from panel (C) were quantified using the Fiji program. Data show mean ± SEM from two experiments in duplicate. **p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. In the figure, the molecular weight of the proteins is indicated at the side of the blots.

FIGURE 3

MSK1 is phosphorylated by p38δ. (A) Recombinant GST-MSK1 (1 µM) was incubated with active recombinant p38α, p38β, p38γ or p38δ for 60 min at 30°C in a phosphorylation reaction mix containing Mg-[γ³²P]-ATP, as described in materials and methods. The activity of recombinant p38α, p38β, p38γ or p38δ was matched using MBP as substrate and 0.5 U/mL were used in the assay. Reaction was stopped with SDS-sample buffer. Samples were resolved in SDS-PAGE and subjected to Coomassie blue staining and autoradiography. (B) Bands corresponding to ³²P-MSK1 were quantified and expressed in arbitrary units (a.u.). Data are shown as mean ± SEM from two experiments in duplicate. (C) Recombinant GST-MSK1 (1 μM) was incubated with p38α or p38δ (0.5 U/mL) as in (A) for the times indicated. Bands corresponding to ³²P-MSK1 were quantified and data represented as mean ± SEM from two experiments in duplicate. (D) GST-p38γKD (1 µM), GST-p38δKD (1 µM) or GST-p38δ(na) (1 µM) were incubated with or without recombinant MSK1 (1 µM) in a phosphorylation reaction mix containing Mg-[γ³²P]-ATP, as described in Figure 1A. ³²P-MSK1 and ³²P-p38 bands were quantified and data represented as mean ± SEM from two experiments in duplicate. (E) GST-p38δKD (1 µM) or GST-p38δ(na) (1 µM) were incubated with or without recombinant MSK1 (1 µM) and BIRB0796 (10 µM) in a phosphorylation reaction mix containing Mg-[γ³²P]-ATP, as described in Figure 1A. ³²P-MSK1 and ³²P-p38δ (GST- and non-GST-tagged) bands were quantified and data represented as mean ± SEM from two experiments in duplicate (right panel). (F) Schematic representation of MSK1 and p38δ phosphorylation in the presence or absence of BIRB0796. (G) GST-p38δKD (1 µM) or GST-p38δ(na) (1 µM) were incubated with or without recombinant MSK1 (1 µM) and SB747651A (100 µM) as described in Figure 1A. ³²P-MSK1 and ³²P-p38δ (GST- and non-GST-tagged) bands were quantified and data represented as mean ± SEM from two experiments in duplicate (right panel). (H) Schematic representation of MSK1 and p38δ phosphorylation in the presence or absence of SB747651A. In the figure, the molecular weight of the proteins is indicated at the side of the blots.

FIGURE 4

MSK1 is activated by p38δ. (A) Recombinant GST-MSK1 (100 ng) was incubated with active recombinant p38δ (0.5 U/mL) for 60 min at 30°C in a phosphorylation reaction mix as described in materials and methods. Reaction was stopped with SDS-sample buffer and samples immunoblotted with the indicated antibodies. MSK1 phosphorylation was detected using the Phospho-MSK1 (Thr581) antibody. Representative immunoblots from two independent experiments in duplicate are shown. (B) Recombinant CREB (1 µg) was incubated with active MSK1 as described in materials and methods. Reaction was stopped with SDS-sample buffer. Samples were resolved in SDS-PAGE and subjected to Coomassie blue staining and autoradiography. Phospho-CREB bands were quantified and data represented in arbitrary units (a.u.) as mean ± SEM from two experiments in duplicate (lower panel). (C) Recombinant GST-CREB (50 ng) was incubated with active recombinant MSK1 as in panel (B) for the indicated times at 30°C in a phosphorylation reaction mix as described in materials and methods. Reaction was stopped with SDS-sample buffer and samples immunoblotted with the Phospho-CREB (Ser133) antibody. Representative immunoblots from two independent experiments are shown. Phospho-CREB bands were quantified and data represented as mean ± SEM from three experiments in duplicate (lower panel). The molecular weight of the proteins is indicated at the side of the blots.

FIGURE 5

Deletion of p38δ impairs the activation of MSK1 downstream targets. (A) BMDM from WT, p38γ^−/−, p38δ^−/− or p38γ/δ^−/− mice were stimulated with 100 ng/mL LPS for 30 min. Cell lysates were immunoblotted with the indicated antibodies. Phospho-CREB (Ser133) antibody also recognises phosphorylated ATF1. Representative immunoblots from three independent experiments in duplicate are shown. (B) WT, p38δ^−/− or p38γ/δ^−/− BMDM were exposed to 100 ng/mL LPS for 1 h with or without 2 µM PD184352, 10 μM SB203580, 3 µM JNK-IN-8 (to inhibit JNKs) or 0.1 µM (to inhibit p38α/β), 1 µM (to inhibit p38α/β and p38γ) or 10 µM (to inhibit p38α/β, p38γ and p38δ) BIRB0796 and then stimulated with LPS. Relative mRNA expression was determined by qPCR for DUSP1. Results were normalized to β-actin mRNA expression and x-fold induction was calculated relative to WT expression at 0 h control. Data show mean ± SEM from one representative experiment of three in triplicate, with similar results. *p ≤ 0.05, **p ≤ 0.001. (C,D) BMDM from WT, p38γ/δ−/− or p38δ−/− mice were stimulated with 100 ng/mL LPS for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. Representative immunoblots from three independent experiments in duplicate are shown. (E) DUSP1 bands from blots shown in panels (C,D) were quantified and data represented in arbitrary units (a.u.) as mean ± SEM from three experiments in duplicate. *p ≤ 0.05; **p ≤ 0.001 relative to WT. (F) WT BMDM were incubated for 1 h with or without 2 µM PD184352, 10 μM SB203580, 3 µM JNK-IN-8 or 0.1, 1 or 10 µM BIRB0796 and then stimulated with LPS. Cell lysates were immunoblotted with the indicated antibodies. Representative immunoblots from two independent experiments in duplicate are shown. DUSP1 bands were quantified and data represented as mean ± SEM from two experiments in duplicate (lower panels). Molecular weight of the proteins is indicated at the side of the blots (G) BMDM were exposed for different times to 100 ng/mL LPS. Relative mRNA expression was determined by qPCR for IL-1Ra. Results were normalized to β-actin mRNA expression and x-fold induction was calculated relative to WT expression at 0 h. Data show mean ± SEM from one representative experiment of two in triplicate, with similar results. *p ≤ 0.05.