Fig. 3

Activation of PPAR-β/δ induced anti-fibrotic responses in TGF-β1-stimulated fibroblasts. A–C, E Control and IPF fibroblasts were serum-starved for 3 h, treated with TGF-β1 (5 ng/ml) for 24 h, followed by the addition of the PPAR-α agonist WY14643 (100 μM, α; A), PPAR-β/δ agonist GW0742 (10 μM, β; A–C, E), and PPAR-γ agonist rosiglitazone (10 μM, γ; A–C, E) either for 24 h (A, C, E) or different time periods (12, 24, 36 and 48 h; B). A PPAR-β/δ activation reversed TGF-β1-induced increase in COL1. Cell lysates were used to detect COL1 and α-SMA by Western blot analysis using GAPDH as reference protein. B, C Reverse of fibrosis phenotype by PPAR-β/δ and PPAR-γ activation was stable for up to 48 h. Cell lysates at 12 to 24 h (B) and 48 h from two other control and IPF patients (C) were used for Western blot analysis using β-actin (β-ACTIN) as reference protein. D Combined activation of PPAR-β/δ and PPAR-γ abolished TGF-β1-induced increase in COL1A2 promoter activity. IPF fibroblasts were transfected with a plasmid containing the luciferase firefly reporter gene adjacent to COL1A2 promoter and Renilla luciferase as second reporter for normalization. At 72 h after transfection, cells were treated with vehicle (Vector) or TGF-β1 (5 ng/ml) for 24 h followed by the addition of the PPAR-β/δ agonist GW0742 (10 μM, β) combined with the PPAR-γ agonist rosiglitazone (10 μM, γ) or vehicle for another 24 h. Cells were lysed and collected for dual luciferase activity measurements. E Ligand activation of PPAR-β/δ together with PPAR-γ strongly decreased the release of collagen produced by TGF-β1-stimulation in control and IPF fibroblasts. Culture media were collected and extracellular collagen was analyzed using Sircol assay. F Combined activation of PPAR-β/δ and PPAR-γ decreased TGF-β1-stimulated release of collagen by control and IPF fibroblasts—this effect was blocked using the respective antagonists. Cells were serum-starved for 3 h, stimulated with vehicle (Control) or TGF-β1 (5 ng/ml) for 24 h, followed by the addition of the PPAR-β/δ agonist GW0742 (10 μM, β) and PPAR-γ agonist rosiglitazone (10 μM, γ) either combined with vehicle or the PPAR-β/δ antagonist GSK0660 (10 nM, β ant) and PPAR-γ antagonist GW9662 (10 μM, γ ant) for another 24 h. Culture media were collected and extracellular collagen was analyzed by Sircol assay