Fig. 4

PPAR-β/δ triggers anti-fibrotic responses by activating MMP-1 in control and IPF fibroblasts. A The transcript of MMP1 is the highest among the different MMPs in control fibroblasts. Analysis of MMP1, MMP2, MMP3, MMP10 and MMP16 of control fibroblasts was done using isolated total RNA and RT-qPCR with HPRT1 as reference gene. B–F Comparative gene expression profile of MMPs was done by RT-qPCR with HPRT1 as reference gene. G PPAR-β/δ attenuated TGF-β1-induced decrease in the amount of active MMP-1. Control and IPF fibroblasts were serum-starved for 3 h, treated with vehicle or TGF-β1 (5 ng/ml) for 24 h, followed by the addition of the PPAR-α agonist WY14643 (100 μM, α), PPAR-β/δ agonist GW0742 (10 μM, β), and PPAR-γ agonist rosiglitazone (10 μM, γ) as well as various combinations thereof for another 24 h. Cell lysates were used to detect active MMP-1 by Western blot analysis using β-actin (β-ACTIN) as reference protein. H Ligand activation of PPAR-β/δ strongly increased the mRNA level of MMP1 in TGF-β1-treated control and IPF fibroblasts. Cells were serum-starved, treated 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) or vehicle for another 24 h. The mRNA levels were measured by RT-qPCR with HPRT1 as reference gene. I Inhibition of MMPs increased TGF-β1-induced release of collagen. Control and IPF fibroblasts were serum-starved for 3 h, treated with vehicle or TGF-β1 (5 ng/ml) for 24 h, followed by the addition of the PPAR-β/δ agonist GW0742 (10 μM, β) and MMP inhibitor (MMP inh., 4-aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamic acid, 20 μM) for another 24 h. The release of collagen into the culture media was measured by Sircol assay