The lysine methyltransferase Smyd1 with its characteristic catalytic SET-domain is highly enriched in the embryonic heart and skeletal muscles, participating in cardiomyogenesis, sarcomere assembly and chromatin remodeling. Recently, significant Smyd1 levels were discovered in endothelial cells (ECs) that responded to inflammatory cytokines. Based on these biochemical properties, we hypothesized that Smyd1 is involved in inflammation-triggered signaling in ECs and therefore, investigated its role within the LPS-induced signaling cascade. Human endothelial cells (HUVECs and EA.hy926 cells) responded to LPS stimulation with higher intrinsic Smyd1 expression. By transfection with expression vectors containing gene inserts encoding either intact Smyd1, a catalytically inactive Smyd1-mutant or Smyd1-specific siRNAs, we show that Smyd1 contributes to LPS-triggered expression and secretion of IL-6 in EA.hy926 cells. Further molecular analysis revealed this process to be based on two signaling pathways: Smyd1 increased the activity of NF-κB and promoted the trimethylation of lysine-4 of histone-3 (H3K4me3) within the IL-6 promoter, as shown by ChIP-RT-qPCR combined with IL-6-promoter-driven luciferase reporter gene assays. In summary, our experimental analysis revealed that LPS-binding to ECs leads to the up-regulation of Smyd1 expression to transduce the signal for IL-6 up-regulation via activation of the established NF-κB pathway as well as via epigenetic trimethylation of H3K4.

NLRP3 inflammasomes are transient large protein aggregates involved in the regulation of the innate immune response but are also associated with endothelial dysfunction during vascular inflammation. While NLRP3 inflammasome assembly and activation is well characterized in immune cells, its role in endothelial cell function remains incompletely understood. This study analyses the function of promyelocytic leukemia (PML) protein, a nuclear scaffold protein that forms so-called PML nuclear bodies (PML-NBs), in the regulation of NLRP3 inflammasome activation in endothelial cell cultures. Following LPS priming and subsequent ATP-induced activation, PML played a dual role: 1. It enhanced NF-kB-dependent transcription of inflammasome components (NLRP3, pro-caspase-1 and pro-IL-1β). 2. At the same time, a post-translational reduction in NLRP3 protein levels and reduced ASC oligomerization were observed, leading to impaired inflammasome activation, as evidenced by lower caspase-1 activity and reduced IL-1β secretion. Proper formation of PML-NBs was critical for this regulatory effect on NLRP3 inflammasome formation, as PML-NBs retained ASC in the nucleus and post-translationally modified NLRP3, presumably affecting its stability. Taken together, these findings suggest that PML represents a regulatory checkpoint in endothelial inflammasome activation, preventing excessive inflammatory responses that could contribute to vascular dysfunction associated with chronic inflammation.