Dataset Information

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. Video Abstract.

SUBMITTER: Robinson KF

PROVIDER: S-EPMC7296971 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Reactive astrogliosis is prominent in most neurodegenerative disorders and is often associated with neuroinflammation. The molecular mechanisms regulating astrocyte-linked neuropathogenesis during injury, aging and human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) are not fully understood. In this study, we investigated the implications of the wingless type (Wnt)/β-catenin signaling pathway in regulating astrocyte function during gliosis. First, we identified that HIV-associated inflammatory cytokines, interleukin (IL)-1β and tumor necrosis factor (TNF)-α induced mediators of the Wnt/β-catenin pathway including β-catenin and lymphoid enhancer-binding factor (LEF)-1 expression in astrocytes. Next, we investigated the regulatory role of β-catenin on primary aspects of reactive astrogliosis, including proliferation, migration and proinflammatory responses, such as IL-6. Knockdown of β-catenin impaired astrocyte proliferation and migration as shown by reduced cyclin-D1 levels, bromodeoxyuridine incorporation and wound healing. HIV-associated cytokines, IL-1β alone and in combination with TNF-α, strongly induced the expression of proinflammatory cytokines including C-C motif chemokine ligand (CCL)2, C-X-C motif chemokine ligand (CXCL)8 and IL-6; however, only IL-6 levels were regulated by β-catenin as demonstrated by knockdown and pharmacological stabilization. In this context, IL-6 levels were negatively regulated by β-catenin. To better understand this relationship, we examined the crossroads between β-catenin and nuclear factor (NF)-κB pathways. While NF-κB expression was significantly increased by IL-1β and TNF-α, NF-κB levels were not affected by β-catenin knockdown. IL-1β treatment significantly increased glycogen synthase kinase (GSK)-3β phosphorylation, which inhibits β-catenin degradation. Further, pharmacological inhibition of GSK-3β increased nuclear translocation of both β-catenin and NF-κB p65 into the nucleus in the absence of any other inflammatory stimuli. HIV+ human astrocytes show increased IL-6, β-catenin and NF-κB expression levels and are interconnected by regulatory associations during HAND. In summary, our study demonstrates that HIV-associated inflammation increases β-catenin pathway mediators to augment activated astrocyte responses including migration and proliferation, while mitigating IL-6 expression. These findings suggest that β-catenin plays an anti-inflammatory role in activated human astrocytes during neuroinflammatory pathologies, such as HAND.

Project description:The canonical Wnt signaling pathway is mediated by interaction of β-catenin with the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors and subsequent transcription activation of Wnt-target genes. In the hematopoietic system, the function of the pathway has been mainly investigated by rather unspecific genetic manipulations of β-catenin that yielded contradictory results. Here, we used a mouse expressing a truncated dominant negative form of the human TCF4 transcription factor (dnTCF4) that specifically abrogates β-catenin-TCF/LEF interaction. Disruption of the β-catenin-TCF/LEF interaction resulted in the accumulation of immature cells and reduced granulocytic differentiation. Mechanistically, dnTCF4 progenitors exhibited downregulation of the Csf3r gene, reduced granulocyte colony-stimulating factor (G-CSF) receptor levels, attenuation of downstream Stat3 phosphorylation after G-CSF treatment, and impaired G-CSF-mediated differentiation. Chromatin immunoprecipitation assays confirmed direct binding of TCF/LEF factors to the promoter and putative enhancer regions of CSF3R. Inhibition of β-catenin signaling compromised activation of the emergency granulopoiesis program, which requires maintenance and expansion of myeloid progenitors. Consequently, dnTCF4 mice were more susceptible to Candida albicans infection and more sensitive to 5-fluorouracil-induced granulocytic regeneration. Importantly, genetic and chemical inhibition of β-catenin-TCF/LEF signaling in human CD34+ cells reduced granulocytic differentiation, whereas its activation enhanced myelopoiesis. Altogether, our data indicate that the β-catenin-TCF/LEF complex directly regulates G-CSF receptor levels, and consequently controls proper differentiation of myeloid progenitors into granulocytes in steady-state and emergency granulopoiesis. Our results uncover a role for the β-catenin signaling pathway in fine tuning the granulocytic production, opening venues for clinical intervention that require enhanced or reduced production of neutrophils.