Abstract
ObjectivesSystemic sclerosis (SSc) is characterised by tissue fibrosis of the major organs of the body including the skin, lungs and heart. We have previously reported that the lncRNA HOTAIR plays a central role in the activation of SSc myofibroblasts, the key cellular elements of fibrosis. HOTAIR induces fibroblast activation through H3K27me3-mediated activation of the Notch signalling pathway. Here we aimed to identify the signalling events downstream of Notch that drive SSc myofibroblast activation.MethodsPatient fibroblasts were obtained from full-thickness forearm skin biopsies of 3 adult patients with SSc of recent onset. The lncRNA HOTAIR was expressed in healthy dermal fibroblasts by lentiviral transduction. Hedgehog signalling pathway was inhibited with GANT61 and GLI2 siRNA. Gamma secretase inhibitors RO4929097 and DAPT were used to block Notch signalling. GSK126 was used to inhibit Enhancer of Zeste 2 (EZH2).ResultsOverexpression of HOTAIR in dermal fibroblasts induced the expression of the Hedgehog pathway transcription factor GLI2. This is mediated by activation of Notch signalling following epigenetic downregulation of miRNA-34a expression. Inhibition of H3K27 methylation and Notch signalling reduced expression of GLI2 in HOTAIR-expressing fibroblasts as well as in SSc dermal fibroblasts. Importantly, the inhibition of GLI2 function using GANT61 or siRNA mitigates the pro-fibrotic phenotype induced by HOTAIR.ConclusionsOur data indicates that GLI2 expression is stably upregulated in SSc myofibroblasts through HOTAIR and that GLI2 mediates the expression of pro-fibrotic markers downstream of Notch.
Highlights
Systemic sclerosis (SSc) is an autoimmune condition that initially presents in the skin of the patient’s hands and feet, where there is a build-up of extracellular matrix resulting in skin fibrosis
Our data indicates that GLI2 expression is stably upregulated in SSc myofibroblasts through HOTAIR and that GLI2 mediates the expression of pro-fibrotic markers downstream of Notch
We show that HOTAIR expression is sufficient to drive Notch-dependent increase of GLI2 expression through Enhancer of Zeste Homology 2 (EZH2)-dependent repression of miRNA-34a and that inhibition of GLI2 is sufficient to reduce the pro-fibrotic phenotype of dermal fibroblasts
Summary
Systemic sclerosis (SSc) is an autoimmune condition that initially presents in the skin of the patient’s hands and feet, where there is a build-up of extracellular matrix resulting in skin fibrosis. The disease progresses to the forearms and legs and in the most severe cases SSc can cause tissue fibrosis in the internal organs (lungs, heart and kidneys). The key role of tissue fibroblasts in the disease has been well-characterised. A number of signalling pathways such as Transforming Growth Factor beta (TGF-β) [1,2,3], Sonic Hedgehog (Shh) [4,5,6] and Notch signalling [7, 8] have been implicated in driving myofibroblast activation, but their precise role and interplay is not fully understood. Epigenetic factors are known to play a key role in the ability of SSc myofibroblasts to maintain their phenotype when explanted from
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