Abstract
BackgroundFibroblasts 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.MethodsLung 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.ResultsWe 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.ConclusionsCEBPA 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.
Highlights
Fibroblasts regulate tissue homeostasis and the balance between tissue repair and fibrosis
Cell culture and adipogenesis assay Primary human lung fibroblasts isolated by explant culture from the lungs of subjects diagnosed with Idiopathic pulmonary fibrosis (IPF) who underwent lung transplantation, or donors whose organs were rejected for transplantation were kindly provided by Peter Bitterman and Craig Henke at the University of Minnesota under a protocol approved by the University of Minnesota Institutional Review Board and by Carol Feghali-Bostwick at the Medical University of South Carolina under a protocol approved by the University of Pittsburgh Institutional Review Board
Across donors we observed a significant decrease in CCAAT/ enhancer-binding protein alpha (CEBPA) transcript levels between fibroblasts derived from lungs of healthy donors versus those derived from subjects with IPF (Fig. 1a)
Summary
Fibroblasts regulate tissue homeostasis and the balance between tissue repair and fibrosis. Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic disease of unknown etiology that is marked by epithelial cell injury, progressive myofibroblast activation, aberrant deposition of extracellular matrix proteins, resulting in failure of the respiratory system and death [1]. Pulmonary lipofibroblasts are located adjacent to alveolar epithelial type II cells (AEC2) in the distal lung, and are recognized by the presence of lipid droplets [5]. Lipofibroblasts are implicated in alveolar maturation and surfactant production [7], as well as epithelial mesenchymal interactions and homeostasis in the lung [8, 9], their presence or absence may play important roles in chronic lung diseases [10]. The mechanisms leading to fibroblast phenotype changes in diseased lung fibroblasts and the functional implications on their fibrogenic activation are not fully understood
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