Prostaglandin E(2) induces fibroblast apoptosis by modulating multiple survival pathways

Abstract

Although the lipid mediator prostaglandin E(2) (PGE(2)) exerts antifibrotic effects by inhibiting multiple fibroblast functions, its ability to regulate fibroblast survival is unknown. Here, we examined the effects of this prostanoid on apoptosis and apoptosis pathways in normal and fibrotic lung fibroblasts. As compared to medium alone, 24 h of treatment with PGE(2) increased apoptosis of normal lung fibroblasts in a dose-dependent manner (EC(50) approximately 50 nM), as measured by annexin V staining, caspase 3 activity, cleavage of poly-ADP-ribose polymerase, and single-stranded DNA levels. PGE(2) also potentiated apoptosis elicited by Fas ligand plus cycloheximide. These proapoptotic actions were dependent on signaling through the EP2/EP4 receptors and by downstream activation of both caspases 8 and 9. Silencing and gene deletion of PTEN demonstrated that the effects of PGE(2) involved decreased activity of the prosurvival molecule Akt. PGE(2) also down-regulated expression of survivin, an inhibitor of apoptosis, and increased expression of Fas. Fibroblasts from patients with pulmonary fibrosis exhibited resistance to the apoptotic effects of PGE(2). These findings show for the first time that, in contrast to its effects on many other cell types, PGE(2) promotes apoptosis in lung fibroblasts through diverse pathways. They provide another dimension by which PGE(2) may inhibit, and perhaps even reverse, fibrogenesis in patients with interstitial lung disease.

Figure 1.

PGE₂ dose-dependently increases fibroblast apoptosis. IMR-90 fibroblasts were treated for 24 h with varying concentrations of PGE₂ and apoptosis was measured by annexin V/PI staining (n=3) (A), caspase 3 activity (n=3) (B), cleavage of PARP (n=4) (C), and ssDNA levels (n=3) (D), as described in Materials and Methods. *P < 0.05 vs. untreated cells.

Figure 2.

PGE₂ potentiates FasL-induced apoptosis of fibroblasts. IMR-90 fibroblasts were treated in the presence or absence of PGE₂ (0.1 μM) and FasL (100 ng/ml) plus CHX (0.5 μg/ml) for 24 h, and cellular apoptosis was measured by annexin V/PI staining (n=3). Results are expressed as the percentage of annexin V⁺/PI⁻ (early apoptosis) cells. *P < 0.05.

Figure 3.

PGE₂ elicits apoptosis via EP2/EP4 signaling. IMR-90 cells were treated for 24 h with PGE₂ (500 nM), the EP2-specific agonist butaprost free acid (BFA; 500 nM), the EP3-specific agonist ONO-AE3–248 (100 nM), or the EP4-specific agonist ONO-AE1–329 (100 nM), and apoptosis was measured by cleavage of PARP. Top panel: representative blot. Bottom panel: densitometric analysis of cleaved PARP relative to α-tubulin (n=3). *P < 0.05 vs. untreated control.

Figure 4.

PGE₂ promotes fibroblast apoptosis via both caspases 8 and 9. A) IMR-90 fibroblasts were treated with PGE₂ (0.5 μM) in the presence or absence of the caspase 8 inhibitor Z-IETD-FMK (25 μM) or the caspase 9 inhibitor Z-LEHD-FMK (25 μM) for 24 h. Caspase 3 activity was measured as described in Materials and Methods; data are expressed as a percentage of activity relative to the no-PGE₂ control (n=4). *P < 0.05. B) IMR-90 fibroblasts were treated with PGE₂ (0.5 μM), Z-IETD-FMK (25 μM), and/or Z-LEHD-FMK (25 μM) for 24 h. Lysates were immunoblotted for cleaved PARP. Top panel: representative immunoblot. Bottom panel: densitometry of cleaved PARP normalized to α-tubulin (n=3).

Figure 5.

Diminished capacity for PGE₂-induced apoptosis in PTEN^−/− fibroblasts. PTEN^−/− and wild-type (wt) murine embryonic fibroblasts were treated for 24 h with PGE₂ at the indicated concentrations. Phosphorylated Akt (A) and cleaved PARP (B) were assayed by immunoblot analysis. Top panels: representative immunoblots. Bottom panels: relative densitometry normalized to untreated controls (wt, n=3; PTEN^−/−, n=4). *P < 0.05.

Figure 6.

PTEN silencing diminishes PGE₂-induced apoptosis in fibroblasts. A) PTEN expression was assayed by immunoblot in cells treated with control or PTEN siRNA for 48 h. Top panel: representative blot. Bottom panel: mean densitometric data (n=4). *P < 0.05. B) PTEN siRNA- and control siRNA-treated cells were exposed to PGE₂ (0.1 μM) for 24 h, and cleaved PARP was assayed by immunoblot. Top panel: representative blot. Bottom panel: densitometry of cleaved PARP/α-tubulin (n=3). CPT (2 μg/ml) was used as a positive control. *P < 0.05 vs. no-PGE₂ treatment.

Figure 7.

PGE₂ modulates fibroblast expression of survivin and Fas receptor. IMR-90 fibroblasts were treated for 24 h with the indicated concentrations of PGE₂, and survivin expression (n=3) (A) and Fas receptor expression (n=3) (B) were determined by immunoblot analysis. *P < 0.05 vs. no-PGE₂ treatment.

Figure 8.

PGE₂-induced apoptosis in adult control and UIP fibroblasts. A) Fibroblasts from the lungs of patients with UIP (n=8, white bars) and nonfibrotic controls (n=3, black bars) were treated with PGE₂ for 24 h, and apoptosis was measured by annexin V/PI staining, with results expressed as the percentage of annexin V⁺/PI⁻ (early apoptosis) cells. Responses of individual UIP lines are displayed at right, segregated within responsive and unresponsive groups. B) UIP (n=3) and nonfibrotic control (n=4) fibroblasts were treated with PGE₂ (0.1 μM) ± FasL (100 ng/ml) + CHX (0.5 μg/ml) for 24 h. Cells were stained for annexin V and PI; percentage of annexin V⁺/PI⁻ cells is shown.

Figure 9.

Schema of PGE₂-induced apoptosis in lung fibroblasts. PGE₂, synthesized by the cyclooxygenase (COX) pathway, induces apoptosis by increasing PTEN activity and decreasing Akt activity. PGE₂ also decreases survivin and increases Fas expression in lung fibroblasts. PGE₂-induced apoptosis is mediated by ligation of the EP2 and EP4 receptors; relative contributions of EP2 and EP4 signaling to the 3 apoptosis pathways remain to be determined.