Targeted regulation of fibroblast state by CRISPR-mediated CEBPA expression

Respiratory Research

Giovanni Ligresti¹  Qi Tan¹  Daniel J. Tschumperlin¹  Aja Aravamudhan¹  Jeffrey A. Meridew¹  Wei Liu² 

[1] Department of Physiology & Biomedical Engineering, Mayo Clinic, Rochester, Mayo Clinic College of Medicine and Science;Emergency Department, First Hospital of China Medical University;

关键词: CEBPA;    Lung fibrosis;    Fibroblast activation;    Adipogenesis;    Lipofibroblast;    CRISPR activation;   

DOI  :  10.1186/s12931-019-1253-1

来源: DOAJ

【 摘 要 】

Abstract Background Fibroblasts regulate tissue homeostasis and the balance between tissue repair and fibrosis. CCAAT/enhancer-binding protein alpha (CEBPA) is a key transcription factor that regulates adipogenesis. CEBPA has been shown to be essential for lung maturation, and deficiency of CEBPA expression leads to abnormal lung architecture. However, its specific role in lung fibroblast regulation and fibrosis has not yet been elucidated. Methods Lung fibroblast CEBPA expression, pro-fibrotic and lipofibroblast gene expression were assessed by qRT-PCR. CEBPA gain and loss of function experiments were carried out to evaluate the role of CEBPA in human lung fibroblast activation with and without TGF-β1 treatment. Adipogenesis assay was used to measure the adiopogenic potential of lung fibroblasts. Finally, CRISPR activation system was used to enhance endogenous CEBPA expression. Results We found that CEBPA gene expression is significantly decreased in IPF-derived fibroblasts compared to normal lung fibroblasts. CEBPA knockdown in normal human lung fibroblasts enhanced fibroblast pro-fibrotic activation and ECM production. CEBPA over-expression by transient transfection in IPF-derived fibroblasts significantly reduced pro-fibrotic gene expression, ECM deposition and αSMA expression and promoted the formation of lipid droplets measured by Oil Red O staining and increased lipofibroblast gene expression. Inhibition of the histone methyl transferase G9a enhanced CEBPA expression, and the anti-fibrotic effects of G9a inhibition were partially mediated by CEBPA expression. Finally, targeted CRISPR-mediated activation of CEBPA resulted in fibroblasts switching from fibrogenic to lipofibroblast states. Conclusions CEBPA expression is reduced in human IPF fibroblasts and its deficiency reduces adipogenic potential and promotes fibrogenic activation. CEBPA expression can be rescued via an inhibitor of epigenetic repression or by targeted CRISPR activation, leading to reduced fibrogenic activation.

【 授权许可】

Unknown