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CBX5/G9a/H3K9me-mediated gene repression is essential to fibroblast activation during lung fibrosis
Giovanni Ligresti, … , Raul A. Urrutia, Daniel J. Tschumperlin
Giovanni Ligresti, … , Raul A. Urrutia, Daniel J. Tschumperlin
Published May 16, 2019
Citation Information: JCI Insight. 2019;4(12):e127111. https://doi.org/10.1172/jci.insight.127111.
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Research Article Cell biology Pulmonology

CBX5/G9a/H3K9me-mediated gene repression is essential to fibroblast activation during lung fibrosis

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Abstract

Pulmonary fibrosis is a devastating disease characterized by accumulation of activated fibroblasts and scarring in the lung. While fibroblast activation in physiological wound repair reverses spontaneously, fibroblast activation in fibrosis is aberrantly sustained. Here we identified histone 3 lysine 9 methylation (H3K9me) as a critical epigenetic modification that sustains fibroblast activation by repressing the transcription of genes essential to returning lung fibroblasts to an inactive state. We show that the histone methyltransferase G9a (EHMT2) and chromobox homolog 5 (CBX5, also known as HP1α), which deposit H3K9me marks and assemble an associated repressor complex, respectively, are essential to initiation and maintenance of fibroblast activation specifically through epigenetic repression of peroxisome proliferator–activated receptor γ coactivator 1 α gene (PPARGC1A, encoding PGC1α). Both TGF-β and increased matrix stiffness potently inhibit PGC1α expression in lung fibroblasts through engagement of the CBX5/G9a pathway. Inhibition of the CBX5/G9a pathway in fibroblasts elevates PGC1α, attenuates TGF-β– and matrix stiffness–promoted H3K9 methylation, and reduces collagen accumulation in the lungs following bleomycin injury. Our results demonstrate that epigenetic silencing mediated by H3K9 methylation is essential for both biochemical and biomechanical fibroblast activation and that targeting this epigenetic pathway may provide therapeutic benefit by returning lung fibroblasts to quiescence.

Authors

Giovanni Ligresti, Nunzia Caporarello, Jeffrey A. Meridew, Dakota L. Jones, Qi Tan, Kyoung Moo Choi, Andrew J. Haak, Aja Aravamudhan, Anja C. Roden, Y.S. Prakash, Gwen Lomberk, Raul A. Urrutia, Daniel J. Tschumperlin

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Figure 1

CBX5 silencing inhibits TGF-β–induced lung fibroblast activation.

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CBX5 silencing inhibits TGF-β–induced lung fibroblast activation.
(A) IM...
(A) IMR90 lung fibroblasts were transfected with CBX5 siRNA for 48 hours followed by TGF-β stimulation for 24 hours. qPCR showed that TGF-β–stimulated profibrotic gene expression was significantly impaired in CBX5-silenced fibroblasts compared with those transfected with control siRNA (n = 3). Data are shown as mean ± SEM of 3 independent experiments performed in duplicate. (*P < 0.05, **P < 0.01, ***P < 0.001, by 1-way ANOVA with Turkey’s multiple comparisons test). (B) Western blotting showing that CBX5 knockdown in lung fibroblasts blocked TGF-β–stimulated αSMA expression (representative blot of n = 3). (C) CBX5-silenced lung fibroblasts displayed deficient ECM protein deposition in response to TGF-β, as demonstrated by the immuno-ECM assay. Both fibronectin and collagen I were significantly reduced in CBX5-silenced fibroblasts compared with control cells. Data shown are representative of 3 independent experiments. Results are expressed as mean ± SEM (***P < 0.001 by 2-tailed, paired t test). Scale bar: 10 μm. (D) Scratch assay shows reduced migratory capacity of CBX5-silenced lung fibroblasts in response to TGF-β compared with control cells. Diminished cell migration was significant at 12 hours and remained impaired at 24 hours following TGF-β exposure. Data represent the mean ± SEM from 1 representative experiment performed in triplicate (**P < 0.01 by 2-tailed, paired t test ). Scale bar: 10 μm. (E) qPCR analysis showing that siRNA knockdown of CBX5 in IPF-derived fibroblasts inhibits TGF-β–induced ACTA2 gene expression (n = 3). Data are shown as mean ± SEM of 3 different IPF cell lines (*P < 0.05, ***P < 0.001, by 1-way ANOVA with Turkey’s multiple comparisons test). (F) ACTA2 gene expression is also significantly reduced in CBX5-silenced IPF fibroblasts in absence of TGF-β (n = 4). Data are shown as mean ± SEM of 4 different IPF cell lines (***P < 0.001 by 2-tailed, paired t test). (G) Schematic representation showing interaction of CBX5 with methylated (me) histone 3 on proximal gene promoters.

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