Dimethyl fumarate alters intracellular calcium handling by redox-mediated pleiotropic effects
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Abstract
Dimethyl fumarate (DMF) is a treatment option for the autoimmune diseases multiple sclerosis and psoriasis. It causes short-term oxidative stress and induces the antioxidant response via the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). Its immunosuppressive effect, however, is poorly understood. As calcium signaling plays a crucial role in the correct functioning of the immune system, this work focuses on understanding how DMF influences the calcium homeostasis in immune cells and if the DMF-induced changes in cellular redox state are responsible for such changes.
I confirmed that DMF evoked short-term oxidative stress in immune cells, which was resolved after 24 h through increased glutathione levels. The major finding of this work was an increase of cytosolic calcium levels in splenocytes immediately after DMF application that remained elevated as a long-term effect. The assumption that the immediate increase of cytosolic calcium was caused by calcium influx through the transient receptor potential ankyrin 1 (TRPA1) channel could not entirely be confirmed. I detected a DMF-caused reduced calcium store content, which could be explained by a reduced expression of the calcium store refilling sarco/endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b). However, SERCA activity was increased due to S-glutathionylation of the redox-regulated cysteine 674. A reduced stromal interaction molecule 1 (STIM1) abundance fits the results that store-operated calcium entry (SOCE) was significantly reduced after DMF treatment. Additionally, oligomerization of STIM1 was found to be modified by reduced glutathione, a hallmark of oxidative stress. STIM1 mutants of the highly conserved cysteines 49 and 56 showed less punctae formation compared to STIM1 wildtype cells when triggered with store depletion. Surprisingly, in contrast to published literature, SOCE was increased in all STIM1 mutants and I did not find any increase in oxidative-stress induced calcium entry. No signs of endoplasmic reticulum (ER) stress were detected, nor changes in glucose uptake, but mitochondrial respiration was altered. The expression of the transcription factors nuclear factor of activated T cells (NFAT) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was not altered, but NFAT translocation was increased.
In summary, DMF causes pleiotropic changes at different levels of the cellular calcium homeostasis, probably due to its ability to redox-modify protein thiols.