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. 2013 May;48(5):550-8.
doi: 10.1165/rcmb.2012-0262OC.

Reversal of myofibroblast differentiation by prostaglandin E(2)

Garth Garrison  1 , Steven K Huang, Katsuhide Okunishi, Jacob P Scott, Loka Raghu Kumar Penke, Anne M Scruggs, Marc Peters-Golden
Affiliations

Reversal of myofibroblast differentiation by prostaglandin E(2)

Garth Garrison et al. Am J Respir Cell Mol Biol. 2013 May.

Abstract

Differentiation of fibroblasts into α-smooth muscle actin (SMA)-expressing myofibroblasts represents a critical step in the pathogenesis of fibrotic disorders, and is generally regarded as irreversible. Prostaglandin E2 (PGE2) has been shown to prevent multiple aspects of fibroblast activation, including the differentiation of fibroblasts to myofibroblasts. Here, we investigated its ability to reverse this differentiated phenotype. Fetal and adult lung fibroblasts were induced to differentiate into myofibroblasts by 24-hour culture with transforming growth factor (TGF)-β1 or endothelin-1. Cells were then treated without or with PGE2 for various intervals and assessed for α-SMA expression. In the absence of PGE2 treatment, α-SMA expression induced by TGF-β1 was persistent and stable for up to 8 days. By contrast, PGE2 treatment effected a dose-dependent decrease in α-SMA and collagen I expression that was observed 2 days after PGE2 addition, peaked at 3 days, and persisted through 8 days in culture. This effect was not explained by an increase in myofibroblast apoptosis, and indeed, reintroduction of TGF-β1 2 days after addition of PGE2 prompted dedifferentiated fibroblasts to re-express α-SMA, indicating redifferentiation to myofibroblasts. This effect of PGE2 was associated with inhibition of focal adhesion kinase signaling, and a focal adhesion kinase inhibitor was also capable of reversing myofibroblast phenotype. These data unambiguously demonstrate reversal of established myofibroblast differentiation. Because many patients have established or even advanced fibrosis by the time they seek medical attention, this capacity of PGE2 has the potential to be harnessed for therapy of late-stage fibrotic disorders.

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Figures

<i>Figure 1.</i>
Figure 1.
Prostaglandin E2 (PGE2) reverses transforming growth factor (TGF)-β1–induced myofibroblast differentiation. (A) IMR-90 cells were pretreated with TGF-β1 (2 ng/ml) for 1 day to induce myofibroblast differentiation, after which medium was removed and cells were treated with or without PGE2 (500 nM) for 1–5 days in serum-free medium. Lysates were collected at the indicated days after treatment and immunoblotted for α-smooth muscle actin (α-SMA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Densitometric values of α-SMA relative to GAPDH, normalized to the Day 1 no-PGE2 control, are shown graphically (n = 3) with a representative immunoblot shown above. *P < 0.05 relative to no-PGE2. (B) CCL-210 fibroblasts were pretreated with TGF-β1 (2 ng/ml) for 1 day to induce myofibroblast differentiation. Cells were then treated with PGE2 (500 nM) or aspirin (ASA) (100 μM) for 1 day before being washed with serum-free medium. Cell lysates were collected at Days 5 and 8 and assayed for α-SMA expression by immunoblot. A representative blot of three experiments is shown. (C) IMR-90 cells were treated with varying doses of PGE2 for 2 days after initial pretreatment with TGF-β1. Expression of α-SMA relative to GAPDH, normalized to no-PGE2 control, is shown (n = 3). (D) IMR-90 cells were treated with or without PGE2 (500 nM) for 2 days after initial pretreatment with TGF-β1. Cells were immunostained for α-SMA; a representative image is shown from three replicates. (E) IMR-90 cells differentiated into myofibroblasts with TGF-b1 were treated with either PGE2 (500 nM), the E prostanoid (EP) 2 agonist, butaprost free acid (500 nM), the EP3 agonist, ONO-AE3-248 (100 nM), the EP4 agonist, ONO-AE1-329 (100 nM), or the direct adenyl cyclase activator, forskolin (100 μM). Expression of α-SMA was assayed by immunoblot (shown above) with densitometry relative to α-tubulin and normalized to no-PGE2 (shown below) (n = 3). Results are expressed as means (±SEM). *P < 0.05 relative to TGF-β1 pretreatment.
<i>Figure 2.</i>
Figure 2.
PGE2 reduces α-SMA mRNA and collagen expression in differentiated myofibroblasts. IMR-90 cells were pretreated with or without TGF-β1 (2 ng/ml) for 1 day to induce myofibroblast differentiation before they were treated with PGE2 (500 nM) for 2 days. α-SMA (A) and collagen 1A1 (B) mRNA were assayed by real-time RT-PCR (n = 6). (C) Cells were pretreated with TGF-β1 (2 ng/ml) before treatment with or without PGE2 (500 nM) for 1–2 days. Collagen I expression was assayed by immunoblotting, with representative immunoblot shown. Densitometric values relative to GAPDH normalized to no-PGE2 Day 1 control are shown below (n = 3). Results are expressed as means ± SEM. *P < 0.05 relative to no-PGE2; #P < 0.05 relative to no–TGF-β1 pretreatment.
<i>Figure 3.</i>
Figure 3.
PGE2 reverses myofibroblast differentiation in the presence of various profibrotic stimuli. (A) IMR-90 cells were pretreated with endothelin-1 (100 ng/ml) for 1 day before treatment with or without PGE2 (500 nM) for 2 days (n = 5). (B) After initial pretreatment with TGF-β1 (2 ng/ml), cells were treated with or without PGE2 in the presence of TGF-β1 (2 ng/ml), and α-SMA was assessed by immunoblot (n = 5). For both (A) and (B), representative immunoblots are shown above, with densitometric values of α-SMA relative to GAPDH and normalized to no-PGE2 treatment shown below. Results are expressed as means ± SEM. *P < 0.05 relative to no-PGE2.
<i>Figure 4.</i>
Figure 4.
PGE2 reverses myofibroblast differentiation in primary adult fibroblasts from nonfibrotic and IPF lung. Primary adult lung fibroblasts from nonfibrotic lungs were pretreated with TGF-β1 (2 ng/ml) for 1 day before treatment with or without PGE2 (500 nM) for 1–2 days. (A) Cell lysates were assayed for α-SMA expression by immunoblot, with representative immunoblot shown above, and densitometric values relative to GAPDH and normalized to no-PGE2 Day 1 control shown below (n = 3). Results are expressed as means ± SEM. *P < 0.05 relative to no-PGE2. (B) α-SMA stress fiber organization was visualized by immunofluorescence microscopy after 2 days of PGE2 treatment, with a representative image of three independent experiments shown. Magnification, 20×. (C) Fibroblasts from two separate patients with IPF were pretreated in the presence or absence of TGF-β1 (2 ng/ml) for 1 day before treatment with or without PGE2 (500 nM) for 2 days. Expression of α-SMA was assayed by immunoblot.
<i>Figure 5.</i>
Figure 5.
PGE2 reduction of α-SMA is not due to increased cellular apoptosis. IMR-90 cells were pretreated with TGF-β1 (2 ng/ml) for 1 day before subsequent treatment with PGE2 (500 nM) for 2 days. Cell counts were performed by hemocytometer (A), and presence of cleaved poly-ADP ribose polymerase (PARP) was assayed by immunoblot (B). A representative immunoblot of three experiments is shown above, with densitometry of cleaved PARP relative to total PARP shown below. FasL (100 ng/ml) and cycloheximide (CHX; 0.5 μg/ml) were administered as a positive control. Results are expressed as means ± SEM.
<i>Figure 6.</i>
Figure 6.
PGE2 reverses myofibroblast differentiation via decreased focal adhesion kinase (FAK) activation. (A) Fibroblasts were pretreated with TGF-β1 (2 ng/ml) for 1 day to induce myofibroblast differentiation before being treated with or without PGE2 (500 nM) for 1–2 days. Expression of active Tyr397-phosphorylated FAK was assayed by immunoblot. A representative immunoblot from IMR-90 cells is shown above, with densitometry of phosphorylated FAK relative to GAPDH shown below (combined result of IMR-90 [n = 3] and CCL-210 [n = 2] cells). *P < 0.05. (B) TGF-β1–induced myofibroblasts were treated with PGE2 for 15 minutes to 6 hours and expression of Tyr397-phosphorylated FAK was assayed by immunoblot. (C) TGF-β1–induced myofibroblasts of fetal and adult origin were treated with the FAK inhibitor, PF573228 (10 μM), for 2 days and expression of collagen I and α-SMA were assayed by immunoblot. A representative immunoblot from IMR-90 cells is shown, with mean densitometry of collagen I and α-SMA expression shown below (combined result of IMR-90 [n = 3] and CCL-210 [n = 2] cells). Results are expressed as means ± SEM. *P < 0.05.
<i>Figure 7.</i>
Figure 7.
Readdition of TGF-β1 to PGE2-dedifferentiated myofibroblasts restores the myofibroblast phenotype. IMR-90 cells differentiated into myofibroblasts with TGF-β1 for 1 day and then dedifferentiated by treatment with PGE2 (500 nM) for 2 days were once again treated with TGF-β1 (2 ng/ml) for 1 day and α-SMA (n = 3) (A) and Tyr397-phosphorylated FAK (B) were assayed by immunoblot. Both a representative immunoblot of α-SMA ([A] top) and densitometric values of α-SMA relative to GAPDH normalized to no-PGE2 control ([A] bottom) are shown. Results are expressed as means ± SEM. *P < 0.05.
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