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

Abstract

Background: 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. Methods: 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. Results: 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. Conclusion: 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.

STAT3 is required for Smo‐dependent signaling and mediates Smo‐targeted treatment resistance and tumorigenesis in Shh medulloblastoma

“…In liver cancer stem cells, Shh/Gli-regulated cell functions were also found to be mediated by activation of the IL-6/STAT3 pathway, but whether there is a similar direct effect of Shh-blockade on IL-6-mediated STAT3 signaling was not reported [59]. Of interest is that in addition to Shh, astrocytes and microglia secrete IL-6 [60,61], and it was reported that treatment with tumors, blockade of STAT3 in our mouse model resulted in significant disruption of Smo-driven MB tumor induction. While blockade of tumor formation did not occur in all mice, it is plausible that tumor initiation in our study may have temporally preceded drug administration and/or drug penetration to the stem-like cell population was not complete in a portion of mice tested.…”

A comprehensive atlas of fetal splicing patterns in the brain of adult myotonic dystrophy type 1 patients

“…TCF3 functions as a transcriptional regulator in neuronal differentiation, with an impact on many other genes ( 68 ). Interestingly, TCF3 can regulate IL-6 signaling, which is disturbed in DM1 ( 69 , 70 ). Since there are differences in splicing aberrations between brain regions in DM1, our findings may not always be representative for other brain regions than the frontal cortex ( 71 ).…”

IL-1β and TNFα Cooperativity in Regulating IL-6 Expression in Adipocytes Depends on CREB Binding and H3K14 Acetylation

“…In this study, we used specific primers directed toward a specific locus within IL-6 proximal promoter, which contains adjacent CRE and NF-IL6 motifs and is located 200 bp upstream from the IL-6 translation initiation site [41,70]. Site directed mutations within CRE or NF-IL6 motifs reduced IL-6 promoter activity in luciferase assays, and eradicated CREB and C/EBPβ bindings in electrophoretic mobility shift assays [65,71], suggesting that these motifs are crucial for IL-6 transcription regulations.…”

“…Studies have been shown that TNFα increases the DNA binding capacity of cyclic AMP Response Element-binding protein (CREB) to CRE-like element (CRE) motif [39], whereas IL-1β enhancing CCAAT/enhancer binding protein beta (C/EBPβ) binds to a consensus site named nuclear factor that specifically binds to an IL1-responsive element in the IL-6 gene (NF-IL6) [40]. Notably, adjacent CRE and NF-IL6 motives are mapped at the IL6 proximal promoter at nucleotides 204-227 upstream from the translation start site (Figure 3A) [41]. Since IL-1β and TNFα cooperatively induced IL-6 transcripts, we examined the ability of CREB and C/EBPβ to bind to the endogenous IL-6 promoter in adipocytes treated with TNFα, IL-1β, alone or in combination, using chromatin immunoprecipitation (ChIP), followed by Q-PCR.…”