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

Abstract

Background/aims: Interleukin-1β (IL-1β) is one of the critical inflammatory factors during atherogenesis. CCAAT/enhancer binding proteins β (C/EBPβ), a regulator of IL-1β production, recently been evidenced as a key player in the development of atherosclerosis. However, the mechanisms of how C/EBPβ regulates the production of IL-1β are unclear. In this study, we aimed to explore the role of C/EBPβ in regulating IL-1β 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 μg/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-1β 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-1β was tested by dual-luciferase reporter assay.

Results: Ox-LDL increased IL-1β production, accompanied with increasing C/EBPβ and p65 expression in a dose- and time-dependent manner. Moreover, C/EBPβ deficiency in macrophages blocked ox-LDL-induced increases in IL-1β expression, maturation as well as p65 activation. However, p65 deficiency inhibited the increase in IL-1β production, but not C/EBPβ expression. Dual-luciferase reporter results showed that overexpression of C/EBPβ significantly enhanced binding activity of p65 to IL-1β promoter. In addition, C/EBP 1β deficiency in macrophages abolished the ox-LDL-induced gene transcription increases of IL-1β, IL-6, p65 and caspase-1.

Conclusions: Our results demonstrate that C/EBPβ acts upstream of NF-κB p65 subunit in ox-LDL-induced IL-1β production in macrophages and may regulate IL-1β maturation by promoting caspase-1. C/EBPβ may be a promising candidate for the prevention and treatment of atherosclerosis.

Keywords: Atherosclerosis; C/EBPβ; IL-1β; Inflammation; NF-κB; Ox-LDL.