Targeted regulation of fibroblast state by CRISPR-mediated CEBPA expression

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.

Keywords: Adipogenesis; CEBPA; CRISPR activation; Fibroblast activation; Lipofibroblast; Lung fibrosis.

Fig. 1

Loss of CEBPA in normal lung fibroblasts enhanced ECM deposition. a) qRT-PCR analysis showing CEBPA expression in IPF-derived fibroblasts (N = 8) and healthy fibroblasts (N = 8). b) Western blotting analysis showing CEBPA protein expression in IPF-derived fibroblasts (N = 5) and healthy fibroblasts (N = 4). c) Western blotting analysis and d) qRT-PCR confirms the knock down of CEBPA expression. e-h) qRT-PCR analysis showing ACTA2, COL1A1, FN1 and CTGF transcript levels in CEBPA knock down normal fibroblasts and their control. i-j) ECM deposition assay shows collagen I and fibronectin production in CEBPA knockdown lung fibroblasts and their control. k) Immunostaining and l) quantification for αSMA expression in both CEBPA knockdown lung fibroblasts and control lung fibroblasts. Scale bar = 100 μm. Data are expressed as mean ± SD (*p < 0.05, ** p < 0.01)

Fig. 2

CEBPA overexpression in IPF-derived fibroblasts reduces their fibrogenic activation. a) qRT-PCR analysis of Cebpa expression in the C/EBPα plasmid-overexpressing IPF fibroblasts compared to empty vector transfected control. b) Western blotting analysis of C/EBPα protein in the C/EBPα-overexpressing IPF fibroblasts compared and empty vector transfected control. c-g) qRT-PCR analysis showing CEBPA, ACTA2, COL1A1, FN1 and CTGF transcript levels in the C/EBPα-overexpressing IPF fibroblasts compared and empty vector transfected control. h-i) ECM deposition assay shows collagen I and fibronectin production in the C/EBPα-overexpressing IPF fibroblasts compared to empty vector transfected control. j-k) Immunostaining for αSMA expression in the C/EBPα-overexpressing IPF fibroblasts compared to empty vector transfected control. Scale bar = 100 μm. l) Western blot analysis of phospho-SMAD2/3 (pSMAD2/3) and total SMAD2/3 and GAPDH protein expression in the C/EBPα-overexpressing IPF fibroblasts and empty vector transfected control with TGF-β for the indicated periods. Time point 1–5: 0 min, 30 min, 60 min, 120 min, 240 min. m) Quantification of the pSMAD2/3 expression to total SMAD2/3 ratio from two independent experiment. Data are expressed as mean ± SD (*p < 0.05, ** p < 0.01)

Fig. 3

CEBPA expression promotes a lipofibroblast phenotype.a-b) Oil Red O staining and quantification in the Cebpa-overexpressing IPF fibroblasts and empty vector transfected control with (AD: adipogenic medium) or without adipogenic medium induction (c: control medium). Scale bar = 100 μm. c-f) qRT-PCR analysis of PLIN2, PPARA, PPARG1 and PPARGC1A transcript levels in Cebpa-overexpressing IPF fibroblasts and empty vector transfected control with or without TGF-β1 treatment for 48 h. Data are expressed as mean ± SD (*p < 0.05, ** p < 0.01, ***p < 0.001, **** p < 0.0001)

Fig. 4

CEBPA expression is enhanced by a G9a inhibitor and partially mediates its anti-fibrotic effects. a) qRT-PCR analysis of CEBPA expression in CEBPA knock down lung fibroblasts and their control with or without BIX01294 for 48 h. b-d) Oil Red O staining and their quantification in IPF fibroblasts with or without BIX01294 in the adipogenic medium for 10 days. e-h) qRT-PCR analysis showing ACTA2, COL1A1, FN1 and CTGF transcript levels in CEBPA knock down lung fibroblasts and their control with or without BIX01294 for 48 h. Data are expressed as mean ± SD (*p < 0.05, ** p < 0.01, ***p < 0.001, **** p < 0.0001)

Fig. 5

CEBPA expression can be restored by CRISPR activation. a) Schematic illustration of the process of CRISPR gene activation (reproduced image is from Horizon Discovery and used with their permission) b) qRT-PCR analysis of CEBPA expression in the dCAS9 expressing IPF fibroblasts with non-targeting gRNA or CEBPA gRNA or CEBPA gRNA&CEBPA siRNA together for 3 days. c-d) Western blot analysis and quantification of Fibronectin and αSMA expression in the dCAS9 expressing IPF fibroblasts with non-targeting gRNA or CEBPA gRNA or CEBPA gRNA&CEBPA siRNA together for 3 days. e-i) qRT-PCR analysis showing ACTA2, COL1A1, FN1, CTGF and PLIN2 transcript levels in the dCAS9 expressing IPF fibroblasts with non-targeting gRNA or CEBPA gRNA or CEBPA gRNA&CEBPA siRNA together for 3 days. Data are expressed as mean ± SD (*p < 0.05, ** p < 0.01, ***p < 0.001, **** p < 0.0001)