Long non-coding RNA HOTAIR induces GLI2 expression through Notch signalling in systemic sclerosis dermal fibroblasts

Abstract

Objectives Systemic 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. Methods Patient 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). Results Overexpression 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. Conclusions 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.

Induction of Pro-Fibrotic CLIC4 in Dermal Fibroblasts by TGF-β/Wnt3a Is Mediated by GLI2 Upregulation

“…Direct interactions between SMAD3 and β-catenin have been identified at numerous promoter sites, [19] including the GLI2 promoter [20]. These findings are of interest in the context of SSc, where both Hh and Wnt-3a signalling are hyper-activated and play important roles in the activation of SSc fibroblasts [21,22].…”

Chloride intracellular channel 4 (CLIC4) expression is transcriptionally regulated by crosstalk of the TGF-β, Wnt and Hedgehog signalling pathways

“…Dysregulated Hh signalling is a characteristic feature of SSc fibroblasts (19). In view of our in vitro data (Figure 1) we hypothesis that GLI transcription factors may contribute to the increased CLIC4 levels observed in SSc fibroblasts.…”

“…Direct interactions between SMAD3 and β-catenin have been identified at numerous promoter sites (17) including the GLI2 promoter (18). These findings are of interest in the context of SSc, where both Shh and Wnt-3a signalling are hyper activated and play important roles in the activation of SSc fibroblasts (19, 20).…”

Epigenetic Changes and Functions in Pneumoconiosis

“…For example, the combination of lncRNA and the chromatin remodeling complex promotes the formation of a dense chromatin structure, thereby generating gene-silencing regions [ 119 ]. lncRNA can also regulate histone modification [ 120 ], promote DNA methylation modification [ 121 ], inhibit transcription factors [ 122 ], and inhibit RNA polymerase II [ 123 ] to regulate mRNA expression, or NATs can directly interfere with mRNA expression, posttranscriptional processing, and transportation [ 112 , 124 , 125 ]. lncRNAs located in the cytoplasm are mainly involved in the regulation and transportation of protein translation.…”