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Basic and translational research
Methyl-CpG-binding protein 2 mediates antifibrotic effects in scleroderma fibroblasts
  1. Ye He1,2,
  2. Pei-Suen Tsou1,
  3. Dinesh Khanna1,
  4. Amr H Sawalha1,3
  1. 1 Division of Rheumatology, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA
  2. 2 Department of Dermatology, The Second Xiangya Hospital, Central South University, Changsha, China
  3. 3 Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, USA
  1. Correspondence to Dr Amr H Sawalha, Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI 48109, USA; asawalha{at}umich.edu

Abstract

Objective Emerging evidence supports a role for epigenetic regulation in the pathogenesis of scleroderma (SSc). We aimed to assess the role of methyl-CpG-binding protein 2 (MeCP2), a key epigenetic regulator, in fibroblast activation and fibrosis in SSc.

Methods Dermal fibroblasts were isolated from patients with diffuse cutaneous SSc (dcSSc) and from healthy controls. MeCP2 expression was measured by qPCR and western blot. Myofibroblast differentiation was evaluated by gel contraction assay in vitro. Fibroblast proliferation was analysed by ki67 immunofluorescence staining. A wound healing assay in vitro was used to determine fibroblast migration rates. RNA-seq was performed with and without MeCP2 knockdown in dcSSc to identify MeCP2-regulated genes. The expression of MeCP2 and its targets were modulated by siRNA or plasmid. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) using anti-MeCP2 antibody was performed to assess MeCP2 binding sites within MeCP2-regulated genes.

Results Elevated expression of MeCP2 was detected in dcSSc fibroblasts compared with normal fibroblasts. Overexpressing MeCP2 in normal fibroblasts suppressed myofibroblast differentiation, fibroblast proliferation and fibroblast migration. RNA-seq in MeCP2-deficient dcSSc fibroblasts identified MeCP2-regulated genes involved in fibrosis, including PLAU, NID2 and ADA. Plasminogen activator urokinase (PLAU) overexpression in dcSSc fibroblasts reduced myofibroblast differentiation and fibroblast migration, while nidogen-2 (NID2) knockdown promoted myofibroblast differentiation and fibroblast migration. Adenosine deaminase (ADA) depletion in dcSSc fibroblasts inhibited cell migration rates. Taken together, antifibrotic effects of MeCP2 were mediated, at least partly, through modulating PLAU, NID2 and ADA. ChIP-seq further showed that MeCP2 directly binds regulatory sequences in NID2 and PLAU gene loci.

Conclusions This study demonstrates a novel role for MeCP2 in skin fibrosis and identifies MeCP2-regulated genes associated with fibroblast migration, myofibroblast differentiation and extracellular matrix degradation, which can be potentially targeted for therapy in SSc.

  • systemic sclerosis
  • fibroblasts
  • autoimmune diseases

https://doi.org/10.1136/annrheumdis-2018-213022

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    Footnotes

    • Handling editor Josef S Smolen

    • Contributors All authors made substantial contributions to the conception or design of the work; or the acquisition, analysis or interpretation of data for the work; drafting the work or revising it critically for important intellectual content; final approval of the version to be published; and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

    • Funding This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health grant number R01AI097134 to Dr Sawalha. Dr Khanna is supported by NIH (National Institute of Arthritis and Musculoskeletal and Skin Diseases) grant number K24AR063120.

    • Competing interests None declared.

    • Patient consent Not required.

    • Ethics approval This study was approved by the institutional review board of the University of Michigan.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Correction notice This article has been corrected since it published Online First. The subtitle ’Gene knockdown experiments' has been updated.