Dynamic regulation of canonical TGFβ signalling by endothelial transcription factor ERG protects from liver fibrogenesis



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The role of the endothelium in protecting from chronic liver disease and TGFβ-mediated fibrosis remains unclear. Here we describe how the endothelial transcription factor ETS-related gene (ERG) promotes liver homoeostasis by controlling canonical TGFβ-SMAD signalling, driving the SMAD1 pathway while repressing SMAD3 activity. Molecular analysis shows that ERG binds to SMAD3, restricting its access to DNA. Ablation of ERG expression results in endothelial-to-mesenchymal transition (EndMT) and spontaneous liver fibrogenesis in EC-specific constitutive hemi-deficient (ErgcEC-Het) and inducible homozygous deficient mice (ErgiEC-KO), in a SMAD3-dependent manner. Acute administration of the TNF-α inhibitor etanercept inhibits carbon tetrachloride (CCL4)-induced fibrogenesis in an ERG-dependent manner in mice. Decreased ERG expression also correlates with EndMT in tissues from patients with end-stage liver fibrosis. These studies identify a pathogenic mechanism where loss of ERG causes endothelial-dependent liver fibrogenesis via regulation of SMAD2/3. Moreover, ERG represents a promising candidate biomarker for assessing EndMT in liver disease.

Chronic liver disease (CLD) is an increasing global health burden; in the UK, liver disease is the fifth biggest cause of mortality with rates doubling from 1991 to 20071. CLD can be triggered by numerous factors including diet, alcohol, viral infection and genetic disorders, which share common features including excessive inflammation2, dysregulated transforming growth factor (TGFβ) signalling3 and dramatic disruption in the vascular architecture of the liver4, 5. Recent studies have revealed that disruption of endothelial cell (EC) homoeostasis can initiate tissue damage and fibrosis6,7,8. Notably, EC have been shown to lose their lineage-specific markers and morphology and acquire a mesenchymal-like phenotype in a process termed endothelial-to-mesenchymal transition (EndMT)9. EndMT is associated with human pathologies such as early vein-graft rejection10 and atherosclerosis11, where it correlates with disease severity. EndMT has been shown to be induced by TGFβ in vitro and in vivo10, 12, and to be enhanced by inflammatory mediators including TNF-α13, 14.

Canonical TGFβ/bone morphogenetic protein (BMP) signalling can activate two opposing signalling cascades, maintaining homoeostasis via phosphorylation of transcription factors SMAD1/5/8, while instigating pro-fibrotic signalling via phosphorylation of SMAD2/315 (Supplementary Fig. 1). In EC, TGFβ isoforms activate SMAD1/5/8 signalling via the receptor ACVRL1 and its co-factor endoglin (ENG)16. BMP ligand-BMP receptor (BMPR) and TGFβ-ACVRL1 interactions selectively induce SMAD1 phosphorylation and directly inhibit TGFβ-ALK5-SMAD3-mediated transcription17. Importantly, TGF-β-induced signalling is influenced by cross-talk with multiple pathways and by lineage-specific co-factors18. Thus, regulation of the balance between SMAD1 and SMAD3 signalling is crucial in maintaining EC homeostasis19, 20.

The ETS transcription factor family plays important roles in vascular development and angiogenesis21. The ETS-related gene (ERG) is the most abundant ETS factor in adult ECs and is essential for endothelial lineage identity; indeed, it is one of only three transcription factors required for reprogramming of progenitors to endothelium22. ERG is crucial for embryonic development and vascular stability23, 24, angiogenesis25 and protection from vascular inflammation26, 27. ERG exerts its pro-homoeostatic, anti-inflammatory function in ECs by driving expression of homoeostatic genes while repressing pro-inflammatory gene expression and inhibiting cytokine-induced EC activation21, 28. In turn, pro-inflammatory molecules TNF-α26 and lipopolysaccharides (LPS)27 induce a significant loss in ERG expression in ECs, suggesting that regulation of ERG expression is key to control of the balance between endothelial homoeostasis and inflammatory signalling.

In this study, we show that ERG maintains the homoeostatic balance of SMAD-dependent signalling in the endothelium, by promoting the SMAD1 pathway while repressing SMAD2/3 activity. We show that loss of ERG in vitro and in vivo results in spontaneous EndMT which is dependent on enhanced SMAD2/3 activity. TNF-α blockade protects ERG expression and reduces phosphorylation of SMAD2/3 in an acute model of liver fibrosis, but is ineffective in ERG hemi-deficient mice. Finally, we show that ERG expression is lost in liver EC from cirrhotic patients with fibrosis related to alcoholic liver disease (ALD) or primary biliary cirrhosis (PBC) and inversely correlates with increased markers of EndMT. Therefore, this study identifies a central role for ERG in regulating EC canonical TGFβ-SMAD signalling to prevent EndMT and ultimately tissue fibrogenesis. Loss of endothelial ERG expression is an early, causative event during tissue fibrogenesis, linked to inflammatory pathways, which can be targeted therapeutically.

Fig. 1
endothelium, Dynamic regulation of canonical TGFβ signalling by endothelial transcription factor ERG protects from liver fibrogenesis

Differentially expression of canonical TGFβ/BMP-SMAD genes in ERG-deficient HUVEC. a Microarray analysis of ERG-dependent genes in HUVEC was performed at 24 and 48 h after ERG depletion, as described (n = 3 biological replicates)24; fold change (log 2) of selected TGFβ/BMP associated genes represented as high (red) and low (blue) expression compared to the median (white). Gene expression data were validated in HUVEC transfected with Control (Con siRNA) or ERG siRNA by b quantitative PCR and c immunoblotting, showing reduction of ERG protein levels following siRNA, d quantification by fluorescence intensity normalised to GAPDH for ERG, SMAD1, ENG, ID1, SMAD3, ALK5 and SMA (n = 5). e Representative images of SMAD1 expression (white; arrows identify expression in the sinusoidal endothelium), VWF (green), SMA (red) and DAPI (blue) in liver tissue from Erg fl/fl and Erg cEC-het mice (Scale bar 50 μm). f Quantitative analysis of TGFβ2 protein expression was performed by ELISA in whole cell HUVEC lysates (n = 7). g Representative image of TGFβ2 expression in HUVEC transfected with control or ERG siRNA by immunofluorescence; nuclei are identified by DRAQ V and cells are co-stained for ERG (green). Scale bar 20 µm. h Representative images of TGFβ2 expression (white; arrows) in large vessel within the liver from Erg fl/fl and Erg cEC-het mice (Scale bar 50 μm). Data were normalised to GAPDH…