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).