Dimethyl Fumarate Targets MSK1, RSK1, 2 and IKKα/β Kinases and Regulates NF-κB /p65 Activation in Psoriasis: A Demonstration of the Effect on Peripheral Blood Mononuclear Cells, Drawn from Two Patients with Severe Psoriasis Before and After Treatment with Dimethyl Fumarate

Abstract

Background: Dimethyl fumarate (DMF) has an inhibitory effect on the production of pro-inflammatory proteins from different cells which participate in the immune reaction in psoriatic skin. Most recently it was shown that DMF is an allosteric covalent inhibitor of the p90 ribosomal S6 kinases (RSK1, 2), determined by X-ray crystallography. DMF binds to a specific cysteine residue in RSK2 and in the closely related mitogen and stress-activated kinases 1 (MSK1) which inhibits further downstream activation.

Objectives: The aim of this study was to review the literature on the effects of DMF on activation of MSK1, RSK1, 2 kinases, and downstream transcription factors NF-κB/p65 and IκBα in cells contributing to the pathogenesis of psoriasis. We also hypothesized and studied if treatment with DMF would inhibit the activation of MSK1, RSK1, 2 kinases in peripheral blood mononuclear cells (PBMCs) in psoriatic patients.

Methods: PBMCs were purified from patients with severe psoriasis before and after 90 days of treatment with DMF. Cells were stimulated with anisomycin, IL-1β or EGF for 10 and 20 minutes. The levels of phosphorylation of MSK1, RSK1, 2 or NF-κB/p65, IκBα were analyzed by Western blotting.

Results: Our case study showed that treatment with DMF inhibited the activation of MSK1 and RSK1, 2 kinases in PBMCs in patients. This supports that DMF is the active metabolite in vivo in psoriatic patients during DMF treatment.

Conclusion: Pro-inflammatory proteins are induced through activation of MSK1 and NF-κB/p65 at (S276). The extracellular signal-regulated kinases (ERK1/2) control cell survival by activating both MSK1 and RSK1, 2 kinases. P-RSK1, 2 activates P-κBα and NF-κB/p65 at (S536). The phosphorylation of NF-κB/p65 at (S276) and (S536) controls different T cell and dendritic cell functions. DMF´s inhibitory effect on MSK1 and RSK1, 2 kinase activations reduces multiple immune reactions in psoriatic patients.

Keywords: 2; DMF; IKKα; IKKβ; IκBα; MSK1; NF-κB/p65; RSK1; psoriasis.

Figure 1

Effect of DMF on PBMCs isolated from psoriatic patient 1 responding to treatment with DMF. PBMCs were collected prior, Day 0 and 90 days after treatments start. (A) PBMCs were stimulated with anisomycin (0.2 µg/ml) or IL-1β (10 ng/ml) for 10 and 20 minutes. In a sample of 20 µg protein/lane the level of immune-staining with antibodies for P-MSK1 (S376), (S212) and total MSK1 or for P-NF-κB p-p65 (S536) and total NF-κB p65 was determined by Western Blotting. The blot was re-incubated with antibodies for β-Actin (B) The level of immune-staining with antibodies for P-RSK1, 2 (S380)/(S386) both, P-RSK2 (S227) and total RSK2 was determined by Western blotting. The blot was re-incubated with antibodies for β-Actin. (C) PBMCs were isolated from healthy individual and stimulated with anisomycin (0.2 µg/ml) or EGF (1 µg/ml) for 0 and 20 minutes. In a sample of 20 µg protein/lane the level of immune-staining with antibodies for P-MSK1 (S376), MSK1 and P-RSK1, 2 (S380)/(S386), RSK1, 2 was determined by Western blotting. The blot was re-incubated with antibodies for β-Actin. The phosphorylated band appears above the total protein.

Figure 2

Effect of DMF on PBMCs isolated from psoriatic patient 1 responding to treatment with DMF. (A) PBMCs were collected prior, Day 0 and Day 90 after treatments start. PBMCs were stimulated as before and in a sample of 20 µg protein/lane the level of immune-staining was tested with rabbit anti-P-IκBα (S32) antibodies. Thereafter the blot was striped and re-incubated with mouse anti-IκBα antibodies to determine the tot amount of IκBα by Western blotting. (Below) The fold induction of IκBα was normalized to day 0 and the non-stimulated PBMCs. (B) The expression level of total Actin was determined by antibodies for Pan-Actin and Western blotting. (Below) The fold induction was normalized to the expression of Pan-Actin at Day 0 and the non-stimulated PBMCs.

Figure 3

Schematic representation in PBMCs of the IL-1β, anisomycin induced activations of MSK1 and RSK1, 2 and IKKα/β kinases via the p38α MAPK and ERK 1/2 signalling pathways 9(A). Effect of DMF on these activations in PBMCs isolated from a patient with severe psoriasis and treated with DMF. DMF inhibited specifically the phosphorylation of MSK1 and RSK1, 2 kinases (B) P-MSK1 mediates the transactivation of P-NF-κB/(S276) while P-RSK1, 2 and P-IKKα mediate the transactivation of P-NF-κB (S536). P-RSK1, 2 and P-IKKβ can both induce the activation of P-IκB (S32). These were all inhibited by treatment with DMF. Inhibition of P-NF-κB/p65 (S276) and (S536) resulted in inhibition of the synthesis of inflammatory proteins and these resulted in the new synthesis of p53/p-p53 (S15). P53 DNA binding repressed BCL-2 and Cyclin D1., Inactivation of P-RSK2 leads also to apoptosis via the cleavage of Caspase-3 and 8 in the cytoplasm.,

Figure 4

Effect of DMF on PBMCs isolated from psoriatic patient 2 not responding to treatment with DMF. PBMCs were isolated Day 0 and Day 90 as before and stimulated with IL-1β (10 ng/ml) or EGF (1 ng/ml) for 10 and 20 minutes. (A) In a sample of 20 µg protein/lane the level of immune-staining was tested with antibodies for P-MSK1 (S376), total MSK1, P-NF-κB p65 (S536) and tot NF-κB p65 were determined by Western blotting. (B) The level of immune-staining with antibodies for P-RSK1, 2 (S380)/(S386) and total RSK2 was determined by Western blotting. The blots were re-probed with antibody for β-Actin.