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

¹ Department of Environmental Medicine, ² Department of Medicine, ³ Lung Biology and Disease Program, ⁴ Department of Immunology and Microbiology, and ⁵ Rochester Eye Institute, University of Rochester, Rochester, New York

Correspondence and requests for reprints should be addressed to Patricia J. Sime, M.D., F.R.C.P., Department of Medicine, University of Rochester, 601 Elmwood Avenue, Box 692, Rochester, NY 14642. E-mail: patricia_sime{at}urmc.rochester.edu

Pulmonary fibrosis is a progressive scarring disease with no effective treatment. Transforming growth factor (TGF)-β 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) is a transcription factor that regulates adipogenesis, insulin sensitization, and inflammation. We report here that a novel PPAR ligand, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), is a potent inhibitor of TGF-β–stimulated differentiation of human lung fibroblasts to myofibroblasts, and suppresses up-regulation of –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 PPAR ligand, 15-deoxy-^12,14-prostaglandin J₂ (15 d-PGJ₂), and 400-fold lower than the synthetic ligand, rosiglitazone. Pharmacologic and genetic approaches were used to demonstrate that CDDO mediates its activity via a PPAR-independent pathway. CDDO and 15 d-PGJ₂ contain an /β unsaturated ketone, which acts as an electrophilic center that can form covalent bonds with cellular proteins. Prostaglandin A₁ and diphenyl diselenide, both strong electrophiles, also inhibit myofibroblast differentiation, but a structural analog of 15 d-PGJ₂ lacking the electrophilic center is much less potent. CDDO does not alter TGF-β–induced Smad or AP-1 signaling, but does inhibit acetylation of CREB binding protein/p300, a critical coactivator in the transcriptional regulation of TGF-β–responsive genes. Overall, these data indicate that certain PPAR ligands, and other small molecules with electrophilic centers, are potent inhibitors of critical TGF-β–mediated profibrogenic activities through pathways independent of PPAR. 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.

Key Words: myofibroblast • peroxisome proliferator–activated receptor • 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid • 15-deoxy-^12,14-prostaglandin J₂ • -smooth muscle actin

CLINICAL RELEVANCE This article describes a novel mechanism by which a peroxisome proliferator–activated receptor agonist blocks myofibroblast differentiation in lung fibroblasts. This has applications for the design of novel treatments for lung fibrosis.