β-Catenin and TCFs/LEF signaling discordantly regulate IL-6 expression in astrocytes

The Wnt/β-catenin signaling pathway is a prolific regulator of cell-to-cell communication and gene expression. Canonical Wnt/β-catenin signaling involves partnering of β-catenin with members of the TCF/LEF family of transcription factors (TCF1, TCF3, TCF4, LEF1) to regulate gene expression. IL-6 is a key cytokine involved in inflammation and is particularly a hallmark of inflammation in the brain. Astrocytes, specialized glial cells in the brain, secrete IL-6. How astrocytes regulate IL-6 expression is not entirely clear, although in other cells NFκB and C/EBP pathways play a role. We evaluated here the interface between β-catenin, TCFs/LEF and C/EBP and NF-κB in relation to IL-6 gene regulation in astrocytes. We performed molecular loss and/or gain of function studies of β-catenin, TCF/LEF, NFκB, and C/EBP to assess IL-6 regulation in human astrocytes. Specifically, siRNA mediated target gene knockdown, cDNA over expression of target gene, and pharmacological agents for regulation of target proteins were used. IL-6 levels was evaluated by real time quantitative PCR and ELISA. We also cloned the IL-6 promoter under a firefly luciferase reporter and used bioinformatics, site directed mutagenesis, and chromatin immunoprecipitation to probe the interaction between β-catenin/TCFs/LEFs and IL-6 promoter activity. β-catenin binds to TCF/LEF to inhibits IL-6 while TCFs/LEF induce IL-6 transcription through interaction with ATF-2/SMADs. β-catenin independent of TCFs/LEF positively regulates C/EBP and NF-κB, which in turn activate IL-6 expression. The IL-6 promoter has two putative regions for TCFs/LEF binding, a proximal site located at -91 nt and a distal site at -948 nt from the transcription start site, both required for TCF/LEF induction of IL-6 independent of β-catenin. IL-6 regulation in human astrocytes engages a discordant interaction between β-catenin and TCF/LEF. These findings are intriguing given that no role for β-catenin nor TCFs/LEF to date is associated with IL-6 regulation and suggest that β-catenin expression in astrocytes is a critical regulator of anti-inflammatory responses and its disruption can potentially mediate persistent neuroinflammation. Video Abstract.