CCAAT/enhancer binding protein β directly regulates the expression of the complement component 3 gene in neural cells: implications for the pro-inflammatory effects of this transcription factor

The CCAAT/enhancer-binding protein β (C/EBPβ) is a transcription factor, which was first identified as a regulator of differentiation and inflammatory processes mainly in adipose tissue and liver; however, its function in the brain was largely unknown for many years. Previous studies from our laboratory indicated that C/EBPβ is implicated in inflammatory process and brain injury, since mice lacking this gene were less susceptible to kainic acid-induced injury.

Altogether, these results indicate that C3 is a downstream target of C/EBPβ, and it could be a mediator of the pro-inflammatory effects of this transcription factor in neural cells.

We first performed cDNA microarrays analysis using hippocampal RNA isolated from C/EBPβ (+/+) and C/EBPβ (-/-) mice. Immunocytochemical and immunohistochemical studies were done to evaluate C/EBPβ and C3 levels. Transient transfection experiments were made to analyze transcriptional regulation of C3 by C/EBPβ. To knockdown C/EBPβ and C3 expression, mouse astrocytes were infected with lentiviral particles expressing an shRNA specific for C/EBPβ or an siRNA specific for C3.

Among the genes displaying significant changes in expression was complement component 3 (C3), which showed a dramatic decrease in mRNA content in the hippocampus of C/EBPβ (-/-) mice. C3 is the central component of the complement and is implicated in different brain disorders. In this work we have found that C/EBPβ regulates C3 levels in rodents glial in vitro and in the rat Substantia nigra pars compacta (SNpc) in vivo following an inflammatory insult. Analysis of the mouse C3 promoter showed that it is directly regulated by C/EBPβ through a C/EBPβ consensus site located at position -616/-599 of the gene. In addition, we show that depletion of C/EBPβ by a specific shRNA results in a significant decrease in the levels of C3 together with a reduction in the increased levels of pro-inflammatory agents elicited by lipopolysaccharide treatment. Conclusions: Altogether, these results indicate that C3 is a downstream target of C/EBPβ, and it could be a mediator of the pro-inflammatory effects of this transcription factor in neural cells.