Long noncoding RNA H19X is a key mediator of TGF-β–driven fibrosis
Elena Pachera, Shervin Assassi, Gloria A. Salazar, Mara Stellato, Florian Renoux, Adam Wunderlin, Przemyslaw Blyszczuk, Robert Lafyatis, Fina Kurreeman, Jeska de Vries-Bouwstra, Tobias Messemaker, Carol A. Feghali-Bostwick, Gerhard Rogler, Wouter T. van Haaften, Gerard Dijkstra, Fiona Oakley, Maurizio Calcagni, Janine Schniering, Britta Maurer, Jörg H.W. Distler, Gabriela Kania, Mojca Frank-Bertoncelj, Oliver Distler
Elena Pachera, Shervin Assassi, Gloria A. Salazar, Mara Stellato, Florian Renoux, Adam Wunderlin, Przemyslaw Blyszczuk, Robert Lafyatis, Fina Kurreeman, Jeska de Vries-Bouwstra, Tobias Messemaker, Carol A. Feghali-Bostwick, Gerhard Rogler, Wouter T. van Haaften, Gerard Dijkstra, Fiona Oakley, Maurizio Calcagni, Janine Schniering, Britta Maurer, Jörg H.W. Distler, Gabriela Kania, Mojca Frank-Bertoncelj, Oliver Distler
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Research Article Autoimmunity Cell biology

Long noncoding RNA H19X is a key mediator of TGF-β–driven fibrosis

Abstract

TGF-β is a master regulator of fibrosis, driving the differentiation of fibroblasts into apoptosis-resistant myofibroblasts and sustaining the production of extracellular matrix (ECM) components. Here, we identified the nuclear long noncoding RNA (lncRNA) H19X as a master regulator of TGF-β–driven tissue fibrosis. H19X was consistently upregulated in a wide variety of human fibrotic tissues and diseases and was strongly induced by TGF-β, particularly in fibroblasts and fibroblast-related cells. Functional experiments following H19X silencing revealed that H19X was an obligatory factor for TGF-β–induced ECM synthesis as well as differentiation and survival of ECM-producing myofibroblasts. We showed that H19X regulates DDIT4L gene expression, specifically interacting with a region upstream of the DDIT4L gene and changing the chromatin accessibility of a DDIT4L enhancer. These events resulted in transcriptional repression of DDIT4L and, in turn, in increased collagen expression and fibrosis. Our results shed light on key effectors of TGF-β–induced ECM remodeling and fibrosis.

Authors

Elena Pachera, Shervin Assassi, Gloria A. Salazar, Mara Stellato, Florian Renoux, Adam Wunderlin, Przemyslaw Blyszczuk, Robert Lafyatis, Fina Kurreeman, Jeska de Vries-Bouwstra, Tobias Messemaker, Carol A. Feghali-Bostwick, Gerhard Rogler, Wouter T. van Haaften, Gerard Dijkstra, Fiona Oakley, Maurizio Calcagni, Janine Schniering, Britta Maurer, Jörg H.W. Distler, Gabriela Kania, Mojca Frank-Bertoncelj, Oliver Distler

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Figure 3

H19X silencing reduces ECM production and myofibroblast activation.

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H19X silencing reduces ECM production and myofibroblast activation. H19X...
H19X was silenced in dermal SSc fibroblasts with 25 nM ASO for a total of 120 hours and stimulated with TGF-β for the last 72 hours in 1% FBS medium. Gene ontology enrichment analysis (GSEA software) performed on significantly downregulated genes (FDR < 0.05 and absolute log2 FC < –0.5) from microarray analysis (A). Heatmap showing significant ECM-related downregulated genes as measured by microarray analysis (FDR < 0.05 and log2 FC > 0.5) (B). The protein levels of fibronectin and αSMA were analyzed by Western blot. Pictures are representative of n = 6–7 biological replicates. The protein level was semiquantified by densitometric analysis. For Western blot, semiquantification fold change was calculated compared with control set as 1 (dashed line) (C and E). The secretion of procollagen1α1 and pan-collagen into supernatants of SSc fibroblasts was quantified with ELISA and Sircol, respectively (D). αSMA and stress fiber formation were assessed by immunofluorescence staining. Scale bar: 50 μm. Pictures are representative of n = 5 biological replicates (F). Cell contraction capacity was evaluated by gel contractility assay. The picture is representative of n = 6 biological replicates (G). Data are presented as single values and mean (n = 5–7). One-way ANOVA (C–E and G). *P < 0.05, **P < 0.01, ***P < 0.001.