C/EBPβ Acts Upstream of NF-κB P65 Subunit in Ox-LDL-Induced IL-1β Production by Macrophages

Background/Aims: Interleukin-1 beta (IL-1 beta) is one of the critical inflammatory factors during atherogenesis. CCAAT/enhancer binding proteins beta (C/EBP beta), a regulator of IL-1 beta production, recently been evidenced as a key player in the development of atherosclerosis. However, the mechanisms of how C/EBP beta regulates the production of IL-1 beta are unclear. In this study, we aimed to explore the role of C/EBP beta in regulating IL-1 beta production in macrophages after oxidized low-density lipoprotein (ox-LDL) exposure and the underlying mechanisms. Methods: RAW264.7 macrophages were treated with 0, 25, 50 or 100 mu g/ml ox-LDL for 12, 24 or 48 h. Small interfering RNAs were used to silence related proteins. The gene and protein expression levels were determined by quantitative real-time polymerase chain reaction or western blot (WB). IL-1 beta secretion was assessed by enzyme-linked immunosorbent assay. The cytoplasmic and nuclear proteins were evaluated by nuclear fractionation followed by WB. Localization of p65 was observed by immunofluorescence. The binding activity of p65 to IL-1 beta was tested by dual-luciferase reporter assay. Results: Ox-LDL increased IL-1 beta production, accompanied with increasing C/EBP beta and p65 expression in a dose-and time-dependent manner. Moreover, C/EBP beta deficiency in macrophages blocked ox-LDL-induced increases in IL-1 beta expression, maturation as well as p65 activation. However, p65 deficiency inhibited the increase in IL-1 beta production, but not C/EBP beta expression. Dual-luciferase reporter results showed that overexpression of C/EBP beta significantly enhanced binding activity of p65 to IL-1 beta promoter. In addition, C/EBP beta deficiency in macrophages abolished the ox-LDL-induced gene transcription increases of IL-1 beta, IL-6, p65 and caspase-1. Conclusions: Our results demonstrate that C/EBP beta acts upstream of NF-kappa B p65 subunit in ox-LDL-induced IL-1 beta production in macrophages and may regulate IL-1 beta maturation by promoting caspase-1. C/EBP beta may be a promising candidate for the prevention and treatment of atherosclerosis.(c) 2018 The Author(s) Published by S. Karger AG, Basel