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. 2021 Mar 22;6(6):e144799.
doi: 10.1172/jci.insight.144799.

Myofibroblast dedifferentiation proceeds via distinct transcriptomic and phenotypic transitions

Sean M Fortier  1 , Loka R Penke  1 , Dana King  2 , Tho X Pham  3 , Giovanni Ligresti  3 , Marc Peters-Golden  1
Affiliations

Myofibroblast dedifferentiation proceeds via distinct transcriptomic and phenotypic transitions

Sean M Fortier et al. JCI Insight. .

Abstract

Myofibroblasts are the major cellular source of collagen, and their accumulation - via differentiation from fibroblasts and resistance to apoptosis - is a hallmark of tissue fibrosis. Clearance of myofibroblasts by dedifferentiation and restoration of apoptosis sensitivity has the potential to reverse fibrosis. Prostaglandin E2 (PGE2) and mitogens such as FGF2 have each been shown to dedifferentiate myofibroblasts, but - to our knowledge - the resultant cellular phenotypes have neither been comprehensively characterized or compared. Here, we show that PGE2 elicited dedifferentiation of human lung myofibroblasts via cAMP/PKA, while FGF2 utilized MEK/ERK. The 2 mediators yielded transitional cells with distinct transcriptomes, with FGF2 promoting but PGE2 inhibiting proliferation and survival. The gene expression pattern in fibroblasts isolated from the lungs of mice undergoing resolution of experimental fibrosis resembled that of myofibroblasts treated with PGE2 in vitro. We conclude that myofibroblast dedifferentiation can proceed via distinct programs exemplified by treatment with PGE2 and FGF2, with dedifferentiation occurring in vivo most closely resembling the former.

Keywords: Apoptosis; Cell Biology; Fibrosis; Pulmonology; Signal transduction.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. PGE2 and FGF2 dedifferentiate established myofibroblasts via distinct signaling pathways.
(A) Experimental scheme depicting myofibroblast differentiation of CCL210 fibroblasts with TGF-β (2 ng/mL) for 48 hours, followed by dedifferentiation with PGE2 (1 μM) or FGF2 (50 ng/mL). (B) αSMA protein expression measured by Western blot analysis 5 days following treatment with PGE2 or FGF2 compared with untreated fibroblast and myofibroblast controls. The histogram depicts mean densitometry values. (C) αSMA stress fibers identified by immunofluorescence microscopy using anti-αSMA antibody and FITC-conjugated secondary antibody. Nuclei are stained with DAPI. (D) Relative ACTA2, COL1A1, and FN1 expression by qPCR in myofibroblasts treated for 24 hours with PGE2 (1 μM), the EP2 agonist butaprost (500 nM), the adenylyl cyclase activator forskolin (500 nM), the PKA specific cAMP analog 6-BNZ cAMP (2 mM), or the Epac specific cAMP analog 8-pCPT cAMP (2 mM). (E) Relative ACTA2, COL1A1, and FN1 expression by qPCR in myofibroblasts treated for 48 hours with FGF2 (50 ng/mL) with and without the MEK/ERK inhibitor UO126 (20 μM). (F) Schematic detailing PGE2 signaling cascade via the EP2 receptor and FGF2 signaling through FGF2R via MEK/ERK. PKA mediates the reduction in ACTA2, COL1A1, and FN1 elicited by PGE2, while MEK/ERK mediates the reduction in ACTA2 and COL1A1 elicited by FGF2. Relative fold changes of indicated genes measured by qPCR are normalized to GAPDH. Data are presented as mean ± SEM. Data points in B represent individual replicate samples from 4 separate experiments. Data points in D and E represent paired replicate samples from 3 experiments. Lines indicate conditions being compared. *P < 0.05, compared with untreated myofibroblast, 1-way ANOVA. Diff, differentiation; De-diff, dedifferentiation; AC, adenylyl cyclase.
Figure 2
Figure 2. RNA-seq of established myofibroblasts treated with or without PGE2 or FGF2.
(A) qPCR analysis of RNA samples submitted for RNA-seq demonstrating approximately 50% reduction in ACTA2 expression in myofibroblasts treated with PGE2 (1 μM) or FGF2 (50 ng/mL) at 6 and 24 hours, respectively. Data points represent paired replicate samples from 3 experiments. Data are presented as mean ± SEM. *P < 0.05, paired 2-tailed t test. (B) Volcano plots representing differential gene expression by log2 fold change (x axis) and adjusted P value (y axis) of total RNA transcripts in PGE2- and FGF2-treated myofibroblasts compared with time-matched controls. (C) Venn diagrams depicting the number of genes differentially expressed as well as those specifically upregulated and downregulated exclusively by PGE2 (red), exclusively by FGF2 (blue), and by both mediators (gray). (D) Principal components analysis of the top 500 variably expressed genes in PGE2- and FGF2-treated myofibroblasts and untreated time-matched controls. Relative fold changes of indicated genes measured by qPCR are normalized to GAPDH. Each colored circle denotes 1 of 3 replicate samples.
Figure 3
Figure 3. Regulation of genes from KEGG pathways enriched by both PGE2 and FGF2.
Heatmap display of individual genes belonging to the specified pathways in myofibroblasts treated with PGE2 or FGF2 for 6 and 24 hours, respectively. Color scale depicts range of log2 fold changes in gene expression.
Figure 4
Figure 4. Gene ontology characteristics of myofibroblasts following treatment with PGE2 or FGF2.
(A and B) Heatmap display of cytoskeletal, ECM-related, and focal adhesion (A) and cell cycle and apoptosis genes (B) in PGE2 - and FGF2-treated myofibroblasts compared with 6- and 24-hour time-matched controls. Color scale depicts range of log2 fold changes in gene expression. ECM, extracellular matrix.
Figure 5
Figure 5. PGE2 and FGF2 modulate the expression of fibrosis-associated long noncoding RNAs and miRNAs in myofibroblasts.
(A and D) Volcano plots representing differential lncRNA expression (A) miRNA expression (D) by log2 fold change (x axis) and adjusted P value (y axis) of total RNA transcripts in PGE2- and FGF2-treated myofibroblasts compared with time-matched controls. Threshold for lncRNAs set by log2 fold change –0.5 to 0.5 and adjusted P < 0.05. Threshold for miRNAs set by adjusted P < 0.05 only. (B and E) Venn diagrams depicting the number of differentially expressed lncRNAs (B) and miRNAs (E) exclusively by PGE2 (red), exclusively by FGF2 (blue), and by both mediators (gray). (C) Heatmap display of fibrosis-associated lncRNAs differentially regulated by PGE2 and/or FGF2. Color scale depicts range of log2 fold changes in gene expression.
Figure 6
Figure 6. Myofibroblasts treated with PGE2 and FGF2 separately or in combination produce distinct cellular morphologies and fibrosis-associated gene expression patterns.
CCL210 fibroblasts were differentiated to myofibroblasts with TGF-β (2 ng/mL) and treated with PGE2 (1 μM), FGF2 (50 ng/mL), or both. (A) Cells were stained with the membrane dye PKH26 (2 μM) and examined by fluorescence microscopy 5 days after treatment. (B) Kinetics of ACTA2 in untreated, PGE2-, FGF2-, and PGE2 + FGF2–treated myofibroblasts. Fibroblasts were treated with TGF-β for 48 hours, followed by treatment and harvesting for mRNA at the indicated time points. (C) Immunofluorescence microscopy and representative Western blot for αSMA in untreated and PGE2 + FGF2–treated myofibroblasts evaluated at 5 days. The histogram depicts mean densitometry values. (D) qPCR analysis of the fibrosis-associated genes ACTA2, COL1A1, FN1, CTGF, VASP, and NOX4 after 24 hours of PGE2 ± FGF2 compared with untreated myofibroblast control. Relative fold changes of indicated genes measured by qPCR are normalized to GAPDH. Data are presented as mean ± SEM; data points represent replicate samples from 3 experiments. Lines indicate conditions being compared. *Statistical significance compared with untreated myofibroblast; +Statistical significance compared with untreated, PGE2-, and FGF2-treated myofibroblasts. *P < 0.05 and +P < 0.05. Performed 2-way ANOVA for B, paired 2-tailed t test for C, and 1-way ANOVA for D. Diff, differentiation; De-diff, dedifferentiation.
Figure 7
Figure 7. PGE2 and FGF2 have opposite effects on myofibroblast proliferation and apoptosis.
(A) CCL210 myofibroblasts were treated with PGE2, FGF2, or PGE2 + FGF2 for 24–72 hours. qPCR analysis of the proliferation gene FOXM1 was performed at 48 hours, while CCNB2, CCND1, and CDKN1C were assessed at 72 hours (top panel). qPCR analysis of the antiapoptotic gene SERPINE1 was performed at 24 hours, while BIRC5 and MYC were assessed at 48 hours; the proapoptotic gene CASP9 was assessed at 72 hours. (B) Proliferation was assessed 96 hours following treatment with PGE2 and/or FGF2 by CyQUANT Cell Proliferation Assay. (C) Apoptosis sensitivity was assessed by measuring total and cleaved CASP3 and PARP by Western blot analysis in myofibroblasts 5 days following addition of PGE2 and/or FGF2, followed by treatment with the death receptor ligand anti-Fas. CASP3 was measured 30 minutes and PARP 1 hour following anti-Fas treatment. Densitometry represents ratio of cleaved products to total protein. Relative fold changes of indicated genes measured by qPCR are normalized to GAPDH. Data are presented as mean ± SEM. Data points represent replicate samples from 3 (A and C) or 4 (B) experiments. Lines indicate conditions being compared. *P < 0.05, compared with untreated myofibroblast; 1-way ANOVA. De-diff, dedifferentiation.
Figure 8
Figure 8. Lung fibroblasts from an in vivo model of fibrosis resolution exhibit similar gene signatures as those determined in myofibroblasts dedifferentiated in vitro.
(A) Experimental scheme for bleomycin-induced pulmonary fibrosis in Col1α1-GFP+ mice with resolving fibrosis (young) and nonresolving fibrosis (aged); mice were sacrificed on day 30, and fibroblasts were flow sorted from lungs and submitted for RNA-seq. (B) Heatmap display of gene expression in mice with resolving fibrosis (compared with the expression in mice with nonresolving fibrosis). Color scale depicts range of log2 fold changes in gene expression. Tnfrsf10b is the mouse homolog of human TNFRSF10A. Gene expression patterns regulated in parallel (blue) or opposite (yellow) to those exhibited with in vitro treatments of human myofibroblasts are indicated in color-filled boxes to the right of the heatmaps.
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