Myofibroblast dedifferentiation proceeds via distinct transcriptomic and phenotypic transitions
Sean M. Fortier, Loka R. Penke, Dana King, Tho X. Pham, Giovanni Ligresti, Marc Peters-Golden
Sean M. Fortier, Loka R. Penke, Dana King, Tho X. Pham, Giovanni Ligresti, Marc Peters-Golden
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Research Article Cell biology Pulmonology

Myofibroblast dedifferentiation proceeds via distinct transcriptomic and phenotypic transitions

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.

Authors

Sean M. Fortier, Loka R. Penke, Dana King, Tho X. Pham, Giovanni Ligresti, Marc Peters-Golden

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Figure 1

PGE2 and FGF2 dedifferentiate established myofibroblasts via distinct signaling pathways.

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PGE2 and FGF2 dedifferentiate established myofibroblasts via distinct si...
(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.