Dimethyl fumarate blocks pro-inflammatory cytokine production via inhibition of TLR induced M1 and K63 ubiquitin chain formation

Abstract

Dimethyl fumarate (DMF) possesses anti-inflammatory properties and is approved for the treatment of psoriasis and multiple sclerosis. While clinically effective, its molecular target has remained elusive - although it is known to activate anti-oxidant pathways. We find that DMF inhibits pro-inflammatory cytokine production in response to TLR agonists independently of the Nrf2-Keap1 anti-oxidant pathway. Instead we show that DMF can inhibit the E2 conjugating enzymes involved in K63 and M1 polyubiquitin chain formation both in vitro and in cells. The formation of K63 and M1 chains is required to link TLR activation to downstream signaling, and consistent with the block in K63 and/or M1 chain formation, DMF inhibits NFκB and ERK1/2 activation, resulting in a loss of pro-inflammatory cytokine production. Together these results reveal a new molecular target for DMF and show that a clinically approved drug inhibits M1 and K63 chain formation in TLR induced signaling complexes. Selective targeting of E2s may therefore be a viable strategy for autoimmunity.

Electrophilic properties of itaconate and derivatives regulate the IκBζ–ATF3 inflammatory axis

“…Proteins were separated on 4–20% polyacrylamide gradient gels (Bio-Rad) and transferred onto PVDF membranes (0.45 µm pore size, Millipore). Non-specific binding was blocked with 5% skim milk (or 5% BSA when phosphoproteins were analysed), and membranes were probed with primary antibodies specific to Nrf2 (#12721), HO-1 (#70081), IκBζ (mouse-specific, #93726), IκBζ (#9244), ATF3 (#D2Y5W), IRAK1 (#4504; sensitivity of IRAK1 detection diminishes upon IRAK1 K63 ubiquitination 26 ), phospho-IKK (Ser176/180, #2697), p62 (also known as SQSTM1) (#5114), phospho-eIF2α (Ser51, #9721), eIF2α (#5324) from Cell Signaling; glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778), IκBα (sc-1643), ATF3 (sc-188), succinate dehydrogenase complex, subunit A (SDHA; sc-166909) from Santa Cruz Biotechnology; NQO1 (ab28947) from Abcam, followed by incubation with anti-rabbit-HRP (1:10,000; sc-2030) or anti-mouse-HRP (1:10,000; sc-2031) from Santa Cruz Biotechnology and Clarity Western ECL substrate (Bio-Rad). Membranes were exposed to X-ray films (Research Products International) and developed using an SRX-101A film processor (Konica Minolta).…”

Transcription Factor NRF2 as a Therapeutic Target for Chronic Diseases: A Systems Medicine Approach

“…Thus, DMF may interact with cysteine residues in several proteins that regulate NF-kB signaling (Blewett et al, 2016). In addition, DMF inhibits ubiquitin-conjugating enzymes and thus prevents the degradation of the IkB repressor of NF-kB in response to IL-1b or Toll-like receptor agonists (McGuire et al, 2016). Moreover, DMF binds directly to specific cysteine residues in protein kinase C-u, a key kinase involved in signaling by the T cell receptor (Blewett et al, 2016).…”

Dimethyl fumarate interferes with MyD88-dependent toll-like receptor signalling pathway in isoproterenol-induced cardiac hypertrophy model

“…The role of TLR activation and its induced inflammatory response in cardiac hypertrophic process are well established . TLR‐dependent transcription and production of pro‐inflammatory cytokines are mainly dependent on the adaptor protein MyD88 . TLR signalling via MyD88‐dependent pathway induces inhibitor of κB (I‐κB) ubiquitination and degradation leading to NF‐κB activation and its nuclear translocation .…”

“…In addition, TLR activates MAPKs signalling pathway, including ERK1/2 and p38 MAPK. MAPK pathways together with NF‐κB are involved in the expression of pro‐inflammatory cytokines and development of cardiac dysfunctions . Moreover, many studies strongly support that ERK1/2‐dependent signalling is enough to produce a hypertrophic phenotype …”

“…McGuire et al . reported that treatment with DMF inhibited NF‐κB and MAPK/ERK 1/2 activation in response to TLR agonist lipopolysaccharide (LPS) in vitro, suggesting its ability to interfere with MyD88‐dependent TLR pathway.…”