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.