Session Type: ACR Abstract Session
Session Time: 2:30PM-4:00PM
Background/Purpose: Fibroblasts explanted from affected tissues in Systemic Sclerosis (SSc), conserve their pro-fibrotic phenotype characterised by increased secretion of collagens and other extracellular matrix proteins and comprise a large proportion of a-Smooth Muscle Actin (a-SMA) positive myofibroblasts. Long non-coding RNAs (lncRNAs) within the HOX loci have been described as master epigenetic regulators within the connective tissue. Specifically, HOTAIR has been shown to drive the specific phenotype of the hands and feet which are also the first body regions affected by SSc. Here we aimed to unravel the mechanisms responsible for the epigenetically stable activation of SSc fibroblasts
Methods: Full thickness skin biopsies were surgically obtained from the forearms oftwelve adult patients with SSc of recent onset. Fibroblasts were isolated and cultured in monolayers and protein and RNA extracted from the fibroblast cultures. Laser capture was performed to isolate cells expressing or not a-SMA as a marker of myofibroblast differentiation. HOTAIR was expressed in healthy dermal fibroblasts by lentiviral induction employing a vector containing the specific sequence. Gamma secretase inhibitors RO4929097 and DAPT were employed to block Notch signalling in SSc fibroblasts. EZH2 was blocked in SSc fibroblasts with the specific inhibitor GSK126.
Results: HOTAIR expression was increased in SSc patients’ skin (100 fold) and in SSc explanted fibroblasts (5 fold). These results were highly significant (p< 0.001 for both). Further, laser captured a-SMA expressing myofibroblasts expressed in average 2.5 fold greater HOTAIR RNA levels compared to a -SMA negative cells from the same donors (P< 0.05). In vitro, we demonstrated that lentiviral induced stable overexpression of HOTAIR in healthy dermal fibroblasts led to their profibrotic activation, including significantly increased expression of Col1A1 and a-SMA both at mRNA and protein levels (2.8 and 1.8 fold respectively, p< 0.05). We further showed that HOTAIR-induced profibrotic activation was due to EZH2 dependent spread of H3k27me3 methylation marker, as demonstrated by complete inhibition by treatment with GSK126. Additionally, we showed that EZH2 led to profibrotic activation by decreasing the expression of miRNA 34a. The reduced miRNA 34a levels in turn led to NOTCH increased expression and signalling pathway activation. Consistent with these findings, treatment of the HOTAIR expressing cells with two different types of gamma secretase inhibitors known to block NOTCH activation, normalised a-SMA expression and suppressed collagen production by 50%. Importantly, treatment with the EZH2 inhibitor GSK126 suppressed collagen production in the HOTAIR expressing cells and SSc fibroblasts by 30% and 50% respectively (p< 0.05) and normalised a-SMA levels in both cell lines to control levels (p< 0.05).
Conclusion: Here we show that the epigenetically stable activation of SSc dermal fibroblasts is due to HOTAIR led EZH2 dependent de-repression of NOTCH signalling. The results of these studies identify a new venue to modulate fibroblasts biology which could inform clinical research to resolve chronic fibrosis and re-establish tissue homeostasis in SSc.
To cite this abstract in AMA style:Wasson C, Abignano G, Ross R, Hermes H, Jimenez S, Del Galdo F. Long Non-coding RNA HOTAIR Induces Myofibroblast Activation in Systemic Sclerosis Through EZH2 Dependent De-repression of NOTCH Signalling Pathway Activation [abstract]. Arthritis Rheumatol. 2019; 71 (suppl 10). https://acrabstracts.org/abstract/long-non-coding-rna-hotair-induces-myofibroblast-activation-in-systemic-sclerosis-through-ezh2-dependent-de-repression-of-notch-signalling-pathway-activation/. Accessed July 13, 2020.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/long-non-coding-rna-hotair-induces-myofibroblast-activation-in-systemic-sclerosis-through-ezh2-dependent-de-repression-of-notch-signalling-pathway-activation/