FIGURE 3.

Involvement of PPARα in fibrate drug-mediated up-regulation of TPP1 mRNA and protein. A–E, mouse primary astrocytes isolated from PPARα^−/− and PPARβ^−/− and wild type mice were treated with different concentrations of gemfibrozil (Gem) and fenofibrate (Feno) in serum-free DMEM/F-12 for 24 h followed by monitoring the mRNA expression of Cln2 by semi-quantitative RT-PCR (A and C) and real time PCR (B and D) and protein level of TPP1 by Western blot (E). F, densitometric analysis of TPP1 levels (relative to β-actin) in PPARα^−/− and PPARβ^−/− and wild type astrocytes by gemfibrozil and fenofibrate treatment. ^a, p <0.05 versus WT control; ^b, p <0.05 versus PPARβ^−/− control; ns, not significant with respect to PPARα^−/− control. G and H, mouse primary astrocytes isolated from WT mice were pretreated with GW9662 (GW) for 30 min followed by treatment with 25 μm gemfibrozil under similar culture conditions. The mRNA expression of Cln2 was monitored by semi-quantitative RT-PCR (G) and real time PCR (H). ^c, p <0.05 versus control; ^d, p <0.05 versus only GW9662-treated. I–K, mouse primary astrocytes isolated from PPARα^−/− and PPARβ^−/− and WT mice were treated with 25 μm gemfibrozil and 10 μm fenofibrate in serum-free DMEM/F-12 for 24 h and double-labeled for TPP1 (red) and GFAP (green) (I, WT; J, PPARα^−/−; K, PPARβ^−/− astrocytes.) DAPI was used to stain nuclei. UN, no treatment. Scale bar, 10 μm. L–N, mouse primary astrocytes isolated from PPARα^−/− and PPARβ^−/−, and WT mice were treated with 25 μm gemfibrozil and 10 μm fenofibrate in serum-free DMEM/F-12 for 24 h. Whole cell extracts containing 5 μg of total protein was incubated at 37 °C with 250 μm 7-amido-4-methylcoumarin in 96-well plates, and readings were taken at an interval of 30, 45, 60, 90, and 120 min. Mean values were taken and plotted in graphical format (L, wild type cells; M, PPARα^−/− cells; N, PPARβ^−/− cells.) All results are mean ± S.E. of at least three independent experiments.