Altered subcellular distribution of MSK1 induced by glucocorticoids contributes to NF-kappaB inhibition

Abstract

Glucocorticoids are widely used anti-inflammatory and immunomodulatory agents, of which the action mechanism is mainly based on interference of hormone-activated glucocorticoid receptor (GR) with the activity of transcription factors, such as nuclear factor-kappaB (NF-kappaB). In addition to the well described interaction-based mutual repression mechanism between the GR and NF-kappaB, additional mechanisms are at play, which help to explain the efficacy of glucocorticoid-mediated gene repression. In this respect, we found that glucocorticoids counteract the recruitment of activated Mitogen- and Stress-activated protein Kinase-1 (MSK1) at inflammatory gene promoters resulting in the inhibition of NF-kappaB p65 transactivation and of concurrent histone H3 phosphorylation. Additionally, we observed that activated GR can trigger redistribution of nuclear MSK1 to the cytoplasm through a CRM1-dependent export mechanism, as a result of an interaction between liganded GR and activated MSK1. These findings unveil a novel aspect within the GR-mediated NF-kappaB-targeting anti-inflammatory mechanism.

Figure 1

Effect of DEX and H89 on TNF-induced histone H3 phosphorylation at the NF-κB-driven gene promoter IL8. A549 cells, serum-starved for 48 h in 0% DMEM, were pretreated with solvent, DEX (1 μM), or H89 (10 μM) for 2 h. Ensuing the indicated stimulation with TNF (2000 IU/ml) for 30 min, cells were lysed and total cell extracts were subjected to ChIP analysis and subsequent qPCR, detecting phosphorylated H3 S10 (H3 S10ph) at the IL8 promoter. qPCR signal of immunoprecipitated IL8 fragments is presented relative to input data. Statistical analysis (ANOVA with Tukey's multiple comparison post-test) was performed to show significant difference with the TNF condition (^*P<0.05). These results are representative for two independent experiments.

Figure 2

Influence of DEX and MSK1 inhibitors on the phosphorylation status and the kinase activity of MSK1 (A) A549 and L929sA cells, serum-starved for 48 h in 0% DMEM, were pretreated with solvent (Solv), DEX (1 μM) or SB203580 (10 μM) and U0126 (10 μM) together (SB/U0), for 2 h. Ensuing the indicated stimulation with TNF (2000 IU/ml), cells were lysed and total cell protein extracts were subjected to western blot analysis detecting human MSK1 S376 or T581 and murine MSK1 S375 and T645 phosphorylations. The displayed bands were detected from one single membrane. (B) L929sA cells, starved for 24 h in 0% DMEM, were pre-incubated for 2 h with DEX (1 μM) or a combination of SB203580 (10 μM) and U0126 (10 μM). After 30 min of TNF (2000 IU/ml) induction, endogenous MSK1 was immunoprecipitated and subjected to an in vitro kinase assay, with a p65 peptide as substrate. (C, D) A549 cells were treated as indicated in (A). Two hours pre-induction with H89 (10 μM) was also included. (C) An immunoblot was set up to detect phosphorylated NF-κB S276. (D) Western blot analysis with a pan α-MSK1 Ab was performed to detect total MSK1 protein. Detection of PARP, NF-κB p65 or aspecific bands served as loading controls. The data shown are representative for three independent experiments.

Figure 3

Recruitment of MSK1 to TNF-activated inflammatory gene promoters. (A, B) Following serum starvation for 48 h, A549 cells were incubated with Solvent (Solv), DEX (1 μM) or H89 (10 μM) for 2 h after which TNF (2000 IU/ml) was added for the indicated time. Crosslinked and sonicated cell lysates were subjected to ChIP analysis against MSK1. qPCR, in triplicate, was used to assay MSK1 recruitment to the IL8 and IL6 gene promoters. MSK1 recruitment on the IL6 and IL8 gene promoters was determined by correction of the qPCR signal of the bound fraction to that of the respective input fraction and presented as % bound/input. Statistical analysis (ANOVA with Tukey's multiple comparison post-test) was performed to show significant difference with the TNF 15′ condition (^*P<0.05; ^***P<0.001) for selected conditions. These data are representative for four independent experiments. (C) A549 cells, serum-starved for 48 h, were incubated with Solv or DEX (1 μM) for 2 h after which TNF (2000 IU/ml) was added for 30 min. The subsequent ChIP procedure against GR was performed as described in (A and B). qPCR of the IL8 promoter region −121/+61 reveals promoter occupancy of GR. Immunoprecipitated bound signal was corrected for input and presented as % bound/input. Statistical analysis (ANOVA with Tukey's multiple comparison post-test) was performed to show significant difference with the Solv condition (^***P<0.001). These data are representative for four independent experiments. The IgG controls indicate aspecific binding.

Figure 4

Indirect immunofluorescence of endogenous MSK1 in A549 cells in the presence of DEX. (A) A549 cells were treated with DEX (1 μM) and/or RU486 (1 μM) and/or TNF (2000 IU/ml) for the indicated times. Through indirect immunofluorescence using an α-MSK1 Ab, endogenous MSK1 (I) was visualized (green) and DAPI staining (II) indicates the nuclei of the cells (blue). (B) ImageJ integrated density analysis of the MSK1 distribution in the cells displayed in (A) is depicted as a scatter dot plot. Statistical analysis (Mann–Whitney U-test) was performed to show the P-value of each condition compared with the Solv condition. These images are representative for six independent experiments.

Figure 5

Immunofluorescence microscopy of EGFP-MSK1, transfected in HEK293T cells. HEK293T cells were transiently transfected with pEGFP-hMSK1 together with empty vector or pSVhGRα. After 24 h serum starvation and subsequent to the indicated inductions with DEX (1 μM) and/or TNF (2000 IU/ml), cells were prepared for immunofluorescence microscopy. Green signal represents EGFP-MSK1 protein (I), whereas blue signal visualizes the Hoechst-stained nuclei (II). Overlays of both pictures are presented in (III). These images are representative for two independent experiments.

Figure 6

Subcellular localization of EGFP-MSK1 and GFP-GRα in HEK293T cells in the presence of LMB. HEK293T cells were transiently transfected with pCMX-hGR-GFP and empty vector (A) or pEGFP-hMSK1 and pSVhGRα (B). Following serum starvation, cells were treated as indicated with LMB (20 ng/ml) and/or DEX (1 μM) and prepared for immunofluorescence analysis. Green signal (I) represents either GFP-GRα (A) or EGFP-MSK1 (B). Hoechst treatment (II), represented by the blue signal, allows imaging of the nuclei.

Figure 7

Indirect immunofluorescence of endogenous MSK1 in A549 cells in the presence of CpdA. (A) A549 cells were treated with solvent (Solv), DEX (1 μM) or CpdA (10 μM) for 60 min. Through indirect immunofluorescence using an anti-MSK1 antibody, endogenous MSK1 (I) was visualized (green) and DAPI staining (II) indicates the nuclei of the cells (blue). In (III), we present an overlay of (I) and (II). (B) ImageJ integrated density analysis of the images in (A) allows us to show MSK1 distribution as a scatter dot plot. Statistical analysis (Mann–Whitney U-test) was performed to show the P-value of each condition compared with the Solv condition. These results are representative of two independent experiments.

Figure 8

Effect of GCs on the interaction of MSK1 and GR. HEK293T cells, transiently transfected with the relevant expression constructs as indicated, were treated with DEX (1 μM) for 2 h. Flag-tagged precipitated complexes were analysed through western blotting for the presence of GR protein. The input fraction of GR, MSK1 and p65 visualizes the transfection efficiency. Background signal was determined through Flag-immunoprecipitation of the irrelevant protein Flag-DRD2S. The displayed bands were blotted onto one single membrane. These results are representative for two independent experiments.

Figure 9

Glucocorticoids target MSK1 to mediate inflammatory gene repression: a hypothetical model. (i) Release of the transcription factor NF-κB from the cytoplasm and its subsequent translocation to the nucleus, followed by DNA binding onto NF-κB-responsive sequences in genes involved in inflammation. (ii) Activation of MAPKs resulting in activation of a fraction of nuclear MSK1, which will in turn phosphorylate NF-κB p65, providing a scaffold for cofactor interaction, and will phosphorylate the H3 S10 residue, relaxing the surrounding chromatin and thus facilitating transcription. (iii) Activation of GR by GCs, leading to GR translocation to the nucleus and its recruitment to the inflammatory gene promoter, and the interaction with p65. (iv) Interaction of activated MSK1 and liganded GR, and CRM1-dependent, GC-instigated nucleocytoplasmic translocation of MSK1 concomitant with loss of MSK1 at the inflammatory gene promoter. Only a fraction of cellular MSK1 is extruded to the cytoplasm. Dissociation of the GR-MSK1-CRM1 complex in the cytoplasm and subsequent return of GR.