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. 2009 Dec;41(6):722-30.
doi: 10.1165/rcmb.2009-0006OC. Epub 2009 Mar 13.

Electrophilic Peroxisome Proliferator-Activated Receptor-Gamma Ligands Have Potent Antifibrotic Effects in Human Lung Fibroblasts

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Electrophilic Peroxisome Proliferator-Activated Receptor-Gamma Ligands Have Potent Antifibrotic Effects in Human Lung Fibroblasts

Heather E Ferguson et al. Am J Respir Cell Mol Biol. .
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Abstract

Pulmonary fibrosis is a progressive scarring disease with no effective treatment. Transforming growth factor (TGF)-beta is up-regulated in fibrotic diseases, where it stimulates differentiation of fibroblasts to myofibroblasts and production of excess extracellular matrix. Peroxisome proliferator-activated receptor (PPAR) gamma is a transcription factor that regulates adipogenesis, insulin sensitization, and inflammation. We report here that a novel PPARgamma ligand, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), is a potent inhibitor of TGF-beta-stimulated differentiation of human lung fibroblasts to myofibroblasts, and suppresses up-regulation of alpha-smooth muscle actin, fibronectin, collagen, and the novel myofibroblast marker, calponin. The inhibitory concentration causing a 50% decrease in aSMA for CDDO was 20-fold lower than the endogenous PPARgamma ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15 d-PGJ(2)), and 400-fold lower than the synthetic ligand, rosiglitazone. Pharmacologic and genetic approaches were used to demonstrate that CDDO mediates its activity via a PPARgamma-independent pathway. CDDO and 15 d-PGJ(2) contain an alpha/beta unsaturated ketone, which acts as an electrophilic center that can form covalent bonds with cellular proteins. Prostaglandin A(1) and diphenyl diselenide, both strong electrophiles, also inhibit myofibroblast differentiation, but a structural analog of 15 d-PGJ(2) lacking the electrophilic center is much less potent. CDDO does not alter TGF-beta-induced Smad or AP-1 signaling, but does inhibit acetylation of CREB binding protein/p300, a critical coactivator in the transcriptional regulation of TGF-beta-responsive genes. Overall, these data indicate that certain PPARgamma ligands, and other small molecules with electrophilic centers, are potent inhibitors of critical TGF-beta-mediated profibrogenic activities through pathways independent of PPARgamma. As the inhibitory concentration causing a 50% decrease in aSMA for CDDO is 400-fold lower than that in rosiglitazone, the translational potential of CDDO for treatment of fibrotic diseases is high.

Figures

Figure 1.
Figure 1.
Both 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) and 15-deoxy-Δ12,14-prostaglandin J2 (15 d-PGJ2) potently suppress transforming growth factor (TGF)-β–induced α–smooth muscle actin (SMA) and calponin expression at very low concentrations, whereas rosiglitazone requires higher concentrations. (A) Primary lung fibroblasts were treated with 5 ng/ml TGF-β for 72 hours with the indicated concentrations of 15 d-PGJ2, CDDO, and rosiglitazone. CDDO potently inhibited α-SMA expression at 10- to 40-fold lower concentrations than 15 d-PGJ2; however, rosiglitazone was only moderately effective at high concentrations. (B) 15 d-PGJ2 (5 μM) and CDDO (1 μM) also inhibit TGF-β–induced expression of the smooth muscle marker, calponin. The experiment was performed three times, each with triplicate samples. The triplicate samples were loaded in adjacent lanes, and representative lanes from a single experiment are shown here. RE, relative expression as determined by densitometry and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (untreated cells = 1). (C) CDDO and 15 d-PGJ2 also inhibit TGF-β–induced accumulation of α-SMA mRNA. Primary human lung fibroblasts were treated with TGF-β alone (5 ng/ml) or with TGF-β and CDDO (1 μM), 15 d-PGJ2 (5 μM), or rosiglitazone (20 μM), and RNA was harvested after 24 hours. CDDO and 15 d-PGJ2, but not rosiglitazone, significantly inhibited TGF-β–stimulated α-SMA message. The results shown are from a single experiment performed in triplicate, and are representative of three independent experiments. *Significant increase compared with untreated; significant decrease compared with TGF-β alone (P < 0.05, ANOVA).
Figure 2.
Figure 2.
Imaging flow cytometry demonstrates that PPARγ ligands attenuate TGF-β–induced myofibroblast differentiation. Human lung fibroblasts were treated with TGF-β in combination with the indicated PPARγ ligands (1 μM CDDO, 5 μM 15 d-PGJ2) for 72 hours, and were then fixed with methanol and stained for α-SMA using a Cy3-conjugated anti–α-SMA antibody, and analyzed on an Amnis Imagestream imaging flow cytometer. Live single cells were identified as described in the online supplemental Materials and Methods. Myofibroblasts were distinguished from fibroblasts based on the intensity (y axis) and fractional area (x axis) of α-SMA staining. The numerical percentage value on each dot-plot refers to the percent of cells contained within the myofibroblast region. Note that TGF-β significantly increases the percentage of myofibroblasts, and this effect is inhibited by both 15 d-PGJ2 and CDDO. See Figure E2 in the online supplement for representative images of cells from both outside and inside the myofibroblast region.
Figure 3.
Figure 3.
CDDO and 15 d-PGJ2 potently inhibit the expression of fibronectin and collagen. (A) Primary lung fibroblasts were treated with 5 ng/ml TGF-β and either 5 μM 15 d-PGJ2 or 1 μM CDDO for 72 hours, and expression of fibronectin was determined by Western blotting. Vinculin, constitutively expressed in fibroblasts, was used as a loading control. RE, relative expression of fibronectin as determined by densitometry and normalized to vinculin (untreated cells = 1). The results shown are representative of three independent experiments, each performed on triplicate wells. (B and C) Primary human lung fibroblasts were treated for 24 hours with TGF-β alone or TGF-β plus CDDO (1μM) or 15 d-PGJ2 (5μM) or rosiglitazone (20 μM). RNA was harvested and analyzed by RT-PCR using collagen I (COL1A1)– and collagen III (COL3A1)–specific primers, and normalized to GAPDH mRNA. CDDO, 15 d-PGJ2, and rosiglitazone all inhibited TGF-β–induced up-regulation of collagen I and III. Results shown are the mean (±SD) for triplicate wells from a single experiment and are representative of three independent experiments (each performed in triplicate). *Significant reduction compared with TGF-β alone (P < 0.05, ANOVA).
Figure 4.
Figure 4.
Neither the irreversible PPARγ antagonist, GW9662, nor overexpression of a dominant-negative PPARγ, inhibits 15 d-PGJ2 and CDDO suppression of α-SMA expression. (A) Primary lung fibroblasts were pretreated with 5 μM GW9662 for 4 hours or left untreated, and were then treated with 5 ng/ml TGF-β and either 5 μM 15 d-PGJ2 or 1 μM CDDO for 72 hours. α-SMA expression was analyzed by Western blot. GW9662 did not restore TGF-β–stimulated expression of α-SMA in cells treated with 15 d-PGJ2 or CDDO. RE, relative expression of α-SMA normalized to GAPDH (untreated cells = 1). Results shown are representative of three independent experiments. (B) Lung fibroblast cultures were infected with a lentiviral vector that coexpresses GFP and a dominant-negative PPARγ (LV-PPARγ-DN, shaded bars), or the GFP-expressing vector only (LV-Empty, solid bars). At 24 hours after infection, the media were changed and TGF-β with or without 5 μM 15 d-PGJ2 or 1 μM CDDO was added to the wells. At 72 hours after treatment, cells were lysed and α-SMA protein levels were analyzed by Western blotting and densitometry. Results shown are the mean (±SE) for two independent experiments with triplicate cultures in each experiment. CDDO and 15 d-PGJ2 significantly reduced TGF-β–driven expression of α-SMA in the presence of both wild-type and DN PPARγ. *Significant reduction compared with TGF-β alone (P < 0.05, ANOVA).
Figure 5.
Figure 5.
Inhibition of TGF-β–stimulated α-SMA expression requires an electrophile. (A) Primary lung fibroblasts were treated with TGF-β and 5 μM 15 d-PGJ2, 5 μM CAY10410, or 1 μM CDDO for 72 hours. Cells were lysed, harvested, and analyzed for α-SMA expression by Western blot. CAY10410 did not inhibit α-SMA expression at the same concentration as 15 d-PGJ2. (B) Primary lung fibroblasts were treated with TGF-β as described in Materials and Methods. With the addition of either 10 μM diphenyl diselenide (DSPS) or 15 μM prostaglandin A1 (PGA1), both potent electrophiles that do not activate PPARγ-dependent transcription. DSPS and PGA1 both inhibited TGF-β–induced α-SMA expression. RE, relative expression of α-SMA normalized to GAPDH (untreated cells = 1). The experiment was performed in triplicate, with the triplicate samples loaded in adjacent lanes of the Western blot. Representative lanes from each condition are shown here.
Figure 6.
Figure 6.
CDDO and 15 d-PGJ2 did not inhibit Smad2/3 phosphorylation, Smad nuclear translocation, or AP-1 signaling. Primary lung fibroblasts were pretreated with 5 μM 15 d-PGJ2 or 1 μM CDDO for 1 hour, followed by treatment with 5 ng/ml TGF-β. Cells were harvested after 15 and 30 minutes and analyzed by Western blot. (A) TGF-β stimulated rapid phosphorylation of Smad 2/3, which was not inhibited by pretreatment with CDDO or 15 d-PGJ2. (B) TGF-β also stimulated translocation of Smad 4 to the nucleus, which was not inhibited by 15 d-PGJ2 or CDDO. Data shown are representative of two independent experiments. PGJ2 indicates 15 d-PGJ2. (C) Primary lung fibroblasts were transfected with an AP-1 luciferase reporter construct and treated with 5 ng/ml TGF-β plus or minus 5 μM 15 d-PGJ2 or 1 μM CDDO for 24 hours, at which time the cells were harvested and luciferase activity was determined. Not only did 15 d-PGJ2 and CDDO not inhibit AP-1 transcriptional activity, they stimulated it. Two independent experiments were performed, each in triplicate. The data shown are means (±SD) for a single representative experiment. *Significant increase over untreated cells; significant increase over TGF-β alone (P < 0.05, ANOVA).
Figure 7.
Figure 7.
CDDO suppresses TGF-β–induced acetylation of CBP/p300. Primary lung fibroblasts were treated with or without TGF-β (5 ng/ml) and 1 μM CDDO. After 72 hours, the cells were harvested, and expression of acetylated CBP/p300 (aCBP/p300) was determined by Western blotting. Levels of aCBP/p300 were determined by densitometry normalized to GAPDH (untreated cells = 1). The data shown are the mean (±SE) for three independent experiments, each performed in triplicate. CDDO significantly inhibited TGF-β–induced acetylation of CBP/p300 (*P < 0.05, ANOVA). Unt, untreated.

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