Abstract
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.
Highlights
Like most other lysine methyltransferases, the members of the Smyd gene family express a characteristic SET domain, which contains the active site required for protein methylation using S-adenosyl methionine (SAM) as a methyl group donor [1,2]
We systematically examined the relationship between LPS, Smyd1 and IL-6 in human endothelial cells (ECs) cultures
Smyd1 vectors or Smyd1-specific siRNAs, we found that LPS induced Smyd1-dependent trimethylation and activation of the IL-6 promoter, suggesting an upstream effect of Smyd1 on IL-6 expression in ECs
Summary
Like most other lysine methyltransferases, the members of the Smyd gene family express a characteristic SET domain, which contains the active site required for protein methylation using S-adenosyl methionine (SAM) as a methyl group donor [1,2]. (1) By the joint action of the two spatially separated SET half domains, four of the five Smyd family members methylate lysines (K) in histones (H) at selected positions, such as H3K4, H3K36 or H4K5 [4,5,6]. The complexity of these histone modifications is increased by the fact that most of the lysines can be methylated once (me1), twice (me2) or even three times (me). According to the histone code [8], methylated histones might be recognized by factors that indirectly trigger changes in the transcription frequency of the downstream target genes [9]
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