Abstract
The innate immune response of pulmonary endothelial cells (EC) to lipopolysaccharide (LPS) induces Forkhead box protein C2 (FOXC2) activation through Toll Like Receptor 4 (TLR4). The mechanisms by which FOXC2 expression is regulated in lung EC under LPS stimulation remain unclear. We postulated that FOXC2 regulates its own expression in sepsis, and its transcriptional autoregulation directs lymphatic EC cell-fate decision. Bioinformatic analysis identified potential FOXC2 binding sites in the FOXC2 promoter. In human lung EC, we verified using chromatin immunoprecipitation (ChIP) and luciferase assays that FOXC2 bound to its own promoter and stimulated its expression after LPS stimulation. Chemical inhibition of histone acetylation by garcinol repressed LPS-induced histone acetylation in the FOXC2 promoter region, and disrupted LPS-mediated FOXC2 binding and transcriptional activation. CRISPR/dCas9/gRNA directed against FOXC2-binding-element (FBE) suppressed LPS-stimulated FOXC2 binding and autoregulation by blocking FBEs in the FOXC2 promoter, and repressed expression of lymphatic EC markers. In a neonatal mouse model of sterile sepsis, LPS-induced FOXC2 binding to FBE and FOXC2 expression in lung EC was attenuated with garcinol treatment. These data reveal a new mechanism of LPS-induced histone acetylation-dependent FOXC2 autoregulation.
Highlights
Forkhead box protein C2 (FOXC2) is a member of the forkhead box (FOX) transcription factor family (Seo et al, 2006)
While our previous study showed that LPS strongly induces FOXC2 in lung and retinal endothelial cells (EC) in vivo and in vitro the mechanisms were not determined (Xia et al, 2018)
In this study we demonstrate that FOXC2 autoregulates its own expression, under native and inflammatory states
Summary
FOXC2 is a member of the forkhead box (FOX) transcription factor family (Seo et al, 2006). It plays a critical role in vascular development. VEGFA activates FOXC2 through kinase insert domain receptor (KDR) pathway in arterial endothelium, and FOXC2 binds to delta like canonical Notch ligand 4 (DLL4) promoter and upregulates DLL4 expression, which in turn activates Notch signaling (Hayashi and Kume, 2008). Hes related family bHLH transcription factor with YRPW motif 1 (HEY1) and HEY2, downstream of DLL4/Notch signaling, collaborating with SRY-box transcription factor 7 (Sox7)/Sox, lead to arterial specification through EphrinB2 activation (Hayashi and Kume, 2008). Unrelated to the role of FOXC2 in developmental signaling, we showed that systemic lipopolysaccharide (LPS)
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