PPAR-γ Ligands Repress TGFβ-Induced Myofibroblast Differentiation by Targeting the PI3K/Akt Pathway: Implications for Therapy of Fibrosis
Figure 2
PPAR-γ ligands inhibit TGFβ-induced phosphorylation of Akt and myofibroblast differentiation in a dose-dependent manner.
A, Primary HLFs were grown until 70–80% confluent, serum starved for 24 hours and treated with the indicated concentrations of PPAR-γ ligands for 48 hours. Total cell lysates were prepared, and subjected to SDS-PAGE followed by immunoblotting. The blot was probed with antibodies against phospho-AktS473, stripped and probed to detect total Akt, αSMA and loading control GAPDH. The relative changes in the ratio of phospho-AktS473/total Akt (R.P.) and relative changes in the expression of αSMA/GAPDH (R.E.) are as indicated in the figure. The experiment was performed in triplicate and a representative blot is shown here. B, LDH release does not increase in response to 15d-PGJ2 or CDDO. Primary human lung fibroblasts were treated with either 5 µM 15d-PGJ2 or 1µM CDDO for 72 hours and LDH release was measured (nmol/min/mL). C, Primary human lung fibroblasts were transfected with a PPRE luciferase reporter and a CMV β-galactosidase construct. Cells were treated with either 5µM 15d-PGJ2 or 1 µM CDDO for 48 hrs and luciferase activity was measured. Background was subtracted and data normalized to β-galactosidase transfection efficiency and reported as fold induction of luciferase units over the untreated samples. These data represent three independent experiments (mean ± S.E. shown, **p≤0.01, *** p≤0.001, compared to untreated).