Peroxisome proliferator-activated receptor-gamma ligands induce heme oxygenase-1 in lung fibroblasts by a PPARgamma-independent, glutathione-dependent mechanism

Abstract

Oxidative stress plays an important role in the pathogenesis of pulmonary fibrosis. Heme oxygenase-1 (HO-1) is a key antioxidant enzyme, and overexpression of HO-1 significantly decreases lung inflammation and fibrosis in animal models. Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a transcription factor that regulates adipogenesis, insulin sensitization, and inflammation. We report here that the PPARgamma ligands 15d-PGJ2 and 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), which have potent antifibrotic effects in vitro, also strongly induce HO-1 expression in primary human lung fibroblasts. Pharmacological and genetic approaches are used to demonstrate that induction of HO-1 is PPARgamma independent. Upregulation of HO-1 coincides with decreased intracellular glutathione (GSH) levels and can be inhibited by N-acetyl cysteine (NAC), a thiol antioxidant and GSH precursor. Upregulation of HO-1 is not inhibited by Trolox, a non-thiol antioxidant, and does not involve the transcription factors AP-1 or Nrf2. CDDO and 15d-PGJ2 contain an alpha/beta unsaturated ketone that acts as an electrophilic center that can form covalent bonds with free reduced thiols. Rosiglitazone, a PPARgamma ligand that lacks an electrophilic center, does not induce HO-1. These data suggest that in human lung fibroblasts, 15d-PGJ2 and CDDO induce HO-1 via a GSH-dependent mechanism involving the formation of covalent bonds between 15d-PGJ2 or CDDO and GSH. Inhibiting HO-1 upregulation with NAC has only a small effect on the antifibrotic properties of 15d-PGJ2 and CDDO in vitro. These results suggest that CDDO and similar electrophilic PPARgamma ligands may have great clinical potential as antifibrotic agents, not only through direct effects on fibroblast differentiation and function, but indirectly by bolstering antioxidant defenses.

Fig. 1.

2-Cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) and 15d-PGJ₂ but not rosiglitazone induce heme oxygenase-1 (HO-1). Primary human lung fibroblasts were treated with the indicated concentrations of CDDO (A) or 15d-PGJ₂ (B) or rosiglitazone. After 24 h, cells were harvested, and HO-1 expression was determined by Western blot. GAPDH was used as a loading control. C: cell lysates were also analyzed for expression of HO-2.

Fig. 2.

Induction of HO-1 by CDDO and 15d-PGJ₂ is peroxisome proliferator-activated receptor-γ (PPARγ) independent. A: primary human lung fibroblasts were pretreated with 5 μM GW9662 for 4 h or left untreated and were then treated with 1 μM CDDO for 24 h. HO-1 expression was determined by Western blot and evaluated by densitometry normalized to GAPDH. RE, relative expression of HO-1 normalized to GAPDH with untreated cells = 1. B: lung fibroblast cultures were infected with a lentiviral vector that coexpresses GFP and a dominant negative PPARγ (LV-PPARγ-DN) or the GFP-expressing vector only (LV-Empty). Twenty-four hours after infection, the media was changed, and CDDO (1 μM), 15d-PGJ₂ (5 μM), or rosiglitazone (20 μM) was added. After a further 24 h, the cells were harvested, and HO-1 expression was determined by Western blot. PPARγ overexpression is demonstrated in cells infected with LV-PPARγ-DN using an antibody to PPARγ (Abcam). Endogenous PPARγ expression is detected in cells infected with the control virus upon longer exposure. RE, relative expression of HO-1 normalized to GAPDH with untreated cells = 1. Results shown are representative of 2 independent experiments, each performed on duplicate wells.

Fig. 3.

CDDO induces HO-1 by a glutathione-dependent mechanism. A: the structures of CDDO and glutathione (GSH) are illustrated. The electrophilic centers of CDDO are circled. CDDO and GSH undergo a Michael addition reaction that results in the formation of a covalent bond between the reduced thiol of GSH and either of the electrophilic centers of CDDO. B: primary human lung fibroblasts were treated with CDDO (1 μM) or 15d-PGJ₂ (5 μM), and intracellular GSH levels were measured 3 and 24 h after treatment. Results shown are representative of 2 independent experiments, each performed in triplicate. *P < 0.05; **P < 0.01, ANOVA. C: primary lung fibroblasts were pretreated for 2 h with 5 mM N-acetyl-l-cysteine (NAC) or 500 μM 6-hydroxyl-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) before addition of 1 μM CDDO. Cells were harvested, and HO-1 was determined by Western blot after 24 h. RE, relative expression of HO-1 normalized to GAPDH with untreated cells = 1. Results shown are representative of 3 independent experiments.

Fig. 4.

Induction of HO-1 by PPARγ ligands is upstream of AP-1 induction. A: primary lung fibroblasts were pretreated with the AP-1 inhibitor SP600125 (0.1 mM) and then treated with CDDO (1 μM), 15d-PGJ₂ (5 μM), or rosiglitazone (20 μM) for 24 h. HO-1 expression was evaluated by Western blot. RE, relative expression of HO-1 normalized to GAPDH with untreated cells = 1. Results shown are representative of 2 independent experiments. B: primary human lung fibroblasts were cotransfected with an AP-1 reporter construct and a β-galactosidase control construct and then treated with CDDO (1 μM), 15d-PGJ₂ (5 μM), or rosiglitazone (20 μM) with or without 5 mM NAC. After 24 h, luciferase activity was determined and normalized to β-galactosidase activity. Results shown are representative of 2 independent experiments, each performed on triplicate cultures. *Significant increase compared with untreated; †significant decrease compared with CDDO alone, P < 0.05 (ANOVA).

Fig. 5.

PPARγ ligands do not induce nuclear translocation of Nrf2 in human lung fibroblasts. Primary human lung fibroblasts were untreated (DMSO only) or were treated with CDDO (1 μM), rosiglitazone (Rosi; 20 μM), or 15d-PGJ₂ (5 μM) for 6 h, and the localization of Nrf2 was determined by immunofluorescence. Nuclei were observed by Hoechst staining (blue). Nrf2 localization in fibroblasts that were untreated (A and B) was primarily cytoplasmic (open arrows). 15d-PGJ₂ induces strong nuclear localization of Nrf2 (solid arrows) (C and D). However, rosiglitazone (E and F) and CDDO (G and H) do not induce nuclear localization, with a broad distribution of NRf2 similar to untreated cells (open arrows). Original magnification, ×400.

Fig. 6.

HO-1 is not directly involved in the inhibition of myofibroblast differentiation by CDDO. Primary lung fibroblasts were pretreated with or without 5 mM NAC and then treated with 5 ng/ml TGF-β and 1 μM CDDO as indicated. After 72 h, protein expression was determined by Western blot. A: representative lanes are shown. B: expression of α-SMA was analyzed by densitometry of triplicate cultures and normalized to GAPDH, with untreated cells = 1. CDDO potently induced HO-1 and inhibited TGF-β-driven α-SMA expression as previously shown. NAC inhibited induction of HO-1 by CDDO, but had only a minor effect on the ability of CDDO to block α-SMA expression. *Significant reduction compared with TGF-β alone and TGF-β + CDDO; †significant increase compared with TGF-β + CDDO, P < 0.05 (ANOVA). Results shown are representative of 2 independent experiments.