C/EBPβ is a key transcription factor of ox-LDL inducing THP-1 cells to release multiple pro-inflammatory cytokines

Abstract

Objective: CCAAT/enhancer binding protein β (C/EBPβ) plays an important role during atherogenesis. However, how C/EBPβ functions remains unclear. In this study, we explore the relationship between C/EBPβ and oxidized LDL-induced multiple pro-inflammatory cytokines released in monocytes.

Materials and methods: THP-1 cells (human monocyte cell line) were stimulated by ox-LDL, ChIP was used to detect the binding function of C/EBPβ to target genes, small interfering RNA was used to knock down the expression of C/EBPβ, Western Blot was used to detect protein expression, and ChIP-seq was used to detect different groups of C/EBPβ bound gene fragments. The integrative genomics viewer (IGV), model-based analysis of ChIP-seq (MACS) were used to visualize the results of ChIP-seq. GO (gene ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes) and Reactome data bases enrichment analysis were performed by the ClusterProfiler software. Ingenuity pathway analysis (IPA) was used to analyze the results of ChIP-seq and to summarize the data within the database.

Results: We identified C/EBPβ as a key protein that regulated IL-1β, IL-6 through database. Then our results confirmed that C/EBPβ could bind directly to the gene of IL-18 and C/EBPβ plays a role in the increased expression and secretion of IL-18 protein after ox-LDL stimulation of THP-1. Using ChIP-seq, we found that the enhanced transcriptional function of C/EBPβ after ox-LDL treatment triggered changes in C/EBPβ-regulated downstream pathways. In the ChIP-seq results, we extracted inflammatory cytokines with significant expression differences, and by comparing them with the database of inflammatory cytokines that C/EBPβ directly regulated, we screened five inflammatory cytokines, CXCL8, IL17B, TNFSF11, CSF3, and CCL2, and the results showed that knockdown of C/EBPβ expression inhibited ox-LDL-induced secretion of CXCL8, TNFSF11, CSF3, and CCL2 by THP-1.

Conclusion: Our results suggest that ox-LDL stimulation enhances C/EBPβ-regulated transcription in THP-1 and C/EBPβ upregulate the release of multiple pro-inflammatory cytokines including IL-18, IL-1β, and IL-6 through direct binding to genes.

Keywords: Atherosclerosis; C/EBPβ; Inflammation; Interleukin-18.